Anti-CD19 Antibodies

ABSTRACT

The present invention provides humanized, chimeric and human anti-CD19 antibodies, anti-CD19 antibody fusion proteins, and fragments thereof that bind to a human B cell marker. Such antibodies, fusion proteins and fragments thereof are useful for the treatment and diagnosis of various B-cell disorders, including B-cell malignancies and autoimmune diseases. In more particular embodiments, the humanized anti-CD19 antibodies may comprise one or more framework region amino acid substitutions designed to improve protein stability, antibody binding and/or expression levels. In a particularly preferred embodiment, the substitutions comprise a Ser91Phe substitution in the hA19 VH sequence.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/680,713, filed Nov. 19, 2012, which was a divisional of U.S. patentapplication Ser. No. 13/398,214 (now U.S. Pat. No. 8,337,840), filedFeb. 16, 2012, which was a divisional of U.S. patent application Ser.No. 12/907,262 (now issued U.S. Pat. No. 8,147,831), filed Oct. 19,2010, which was a divisional of U.S. patent application Ser. No.12/266,999 (now issued U.S. Pat. No. 7,902,338), filed Nov. 7, 2008,which was a continuation-in-part of U.S. patent application Ser. No.11/445,410 (now issued U.S. Pat. No. 7,462,352), filed Jun. 1, 2006,which was a divisional of U.S. patent application Ser. No. 10/903,858(now issued U.S. Pat. No. 7,109,304), filed Aug. 2, 2004, which claimedpriority to a provisional U.S. Patent Application No. 60/491,282, filedJul. 31, 2003, the contents of each of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to anti-CD19 antibodies, particularlyhumanized, chimeric and human anti-CD19 antibodies, particularlymonoclonal antibodies (MAbs) and fragments thereof, either naked orconjugated to at least one therapeutic and/or diagnostic agent, andmethods of use thereof. In particular, the anti-CD19 antibodies can beused for treating B cell disease such as, for example, a malignancy, aninflammatory disease or disorder, or an autoimmune disease. In moreparticular embodiments, the anti-CD19 antibodies may comprise one ormore substituted amino acids designed to optimize a physical and/orphysiological characteristic of the antibody.

2. Description of Related Art

The immune system of vertebrates consists of a number of organs and celltypes which have evolved to accurately recognize foreign antigens,specifically bind to, and eliminate/destroy such foreign antigens.Lymphocytes, among other cell types, are critical to the immune system.Lymphocytes are divided into two major sub-populations, T cells and Bcells. Although inter-dependent, T cells are largely responsible forcell-mediated immunity and B cells are largely responsible for antibodyproduction (humoral immunity).

In humans, each B cell can produce an enormous number of antibodymolecules. Such antibody production typically ceases (or substantiallydecreases) when a foreign antigen has been neutralized. Occasionally,however, proliferation of a particular B cell will continue unabated andmay result in a cancer known as a B cell lymphoma or leukemia. B-celllymphomas, such as the B-cell subtype of non-Hodgkin's lymphoma, aresignificant contributors to cancer mortality.

The response of B-cell malignancies to various forms of treatment ismixed. For example, in cases in which adequate clinical staging ofnon-Hodgkin's lymphoma is possible, field radiation therapy can providesatisfactory treatment. Still, about one-half of the patients die fromthe disease. Devesa et al., J. Nat'l Cancer Inst. 79:701 (1987).

The majority of chronic lymphocytic leukemias are of the B-cell lineage.Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990). This type ofB-cell malignancy is the most common leukemia in the Western world.Goodman et al., Leukemia and Lymphoma 22: 1 (1996). The natural historyof chronic lymphocytic leukemia falls into several phases. In the earlyphase, chronic lymphocytic leukemia is an indolent disease,characterized by the accumulation of small maturefunctionally-incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment canprovide symptomatic relief, the overall survival of the patients is onlyminimally affected. The late stages of chronic lymphocytic leukemia arecharacterized by significant anemia and/or thrombocytopenia. At thispoint, the median survival is less than two years. Foon et al., AnnalsInt. Medicine 113:525 (1990). Due to the very low rate of cellularproliferation, chronic lymphocytic leukemia is resistant to cytotoxicdrug treatment. Traditional methods of treating B-cell malignancies,including chemotherapy and radiotherapy, have limited utility due totoxic side effects.

B cells comprise cell surface proteins which can be utilized as markersfor differentiation and identification. One such human B-cell marker isa CD19 antigen and is found on mature B cells but not on plasma cells.CD19 is expressed during early pre-B cell development and remains untilplasma cell differentiation. CD19 is expressed on both normal B cellsand malignant B cells whose abnormal growth can lead to B-celllymphomas. For example, CD19 is expressed on B-cell lineagemalignancies, including, but not limited to non-Hodgkin's lymphoma,chronic lymphocytic leukemia, and acute lymphoblastic leukemia.

A potential problem with using non-human monoclonal antibodies (e.g.,murine monoclonal antibodies) is typically lack of human effectorfunctionality. In other words, such antibodies may be unable to mediatecomplement-dependent lysis or lyse human target cells throughantibody-dependent cellular toxicity or Fc-receptor mediatedphagocytosis. Furthermore, non-human monoclonal antibodies can berecognized by the human host as a foreign protein and, therefore,repeated injections of such foreign antibodies can lead to the inductionof immune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response.

The use of chimeric antibodies is more preferred because they do notelicit as strong a HAMA response as murine antibodies. Chimericantibodies are antibodies which comprise portions from two or moredifferent species. For example, Liu, A. Y. et al, “Production of aMouse-Human Chimeric Monoclonal Antibody to CD20 with PotentFc-Dependent Biologic Activity” J. Immun. 139/10:3521-3526 (1987),describe a mouse/human chimeric antibody directed against the CD20antigen. See also, PCT Publication No. WO 88/04936. However, noinformation is provided as to the ability, efficacy or practicality ofusing such chimeric antibodies for the treatment of B cell disorders inthe reference. It is noted that in vitro functional assays (e.g.,complement-dependent lysis (CDC); antibody dependent cellularcytotoxicity (ADCC), etc.) cannot inherently predict the in vivocapability of a chimeric antibody to destroy or deplete target cellsexpressing the specific antigen. See, for example, Robinson, R. D. etal., “Chimeric mouse-human anti-carcinoma antibodies that mediatedifferent anti-tumor cell biological activities,” Hum. Antibod.Hybridomas 2:84-93 (1991) (chimeric mouse-human antibody havingundetectable ADCC activity). Therefore, the potential therapeuticefficacy of a chimeric antibody can only truly be assessed by in vivoexperimentation, preferably in the species of interest for the specifictherapy.

One approach that has improved the ability of murine monoclonalantibodies to be effective in the treatment of B-cell disorders has beento conjugate a radioactive label or chemotherapeutic agent to theantibody, such that the label or agent is localized at the tumor site.For example, studies indicate that ⁹⁰Y labeled anti-CD19 antibodies canbe used to reduce lymphoma in mice (McDevitt et al., Leukemia 16:60,2002), anti-CD19 antibodies conjugated to idarubicin result in tumorregression in an experimental model (Rowland et al., Cancer Immunol.Immunother., 37:195, 1993), and ¹²⁵I and ¹¹¹In radiolabeled anti-CD19 isspecifically taken up in tumor bearing organs (Mitchell et al., J. Nucl.Med., 44: 1105, 2003). Combination therapy with an anti-CD19 antibody isalso disclosed in Ek et al., Leuk. Lymphoma 31: 143 (1998) and Uckun etal., Blood, 79:3116 (1992). Treatment of human B cell lymphoma with ananti-CD19 antibody and anti-CD3 x anti-CD19 diabody is disclosed inHekman et al., Cancer Immunol. Immunother., 32:364 (1991) and Cochloviuset al., J. Immunol., 165:888 (2000), respectively.

However, these approaches have not eliminated the obstacles associatedwith using murine antibodies, despite the fact that many patients withlymphoma who have received prior aggressive cytotoxic chemotherapy areimmune suppressed, thus having lower HAMA rates than lymphoma patientswho have not been heavily pretreated.

Inflammatory diseases, including autoimmune diseases are also a class ofdiseases associated with B-cell disorders. The most common treatmentsare corticosteroids and cytotoxic drugs, which can be very toxic. Thesedrugs also suppress the entire immune system, can result in seriousinfection, and have adverse affects on the bone marrow, liver andkidneys. Other therapeutics that have been used to treat Class IIIautoimmune diseases have been directed against T-cells and macrophages.There is a need for more effective methods of treating autoimmunediseases, particularly Class III autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention provides humanized, chimeric and human anti-CD19monoclonal antibodies and fragments thereof, and antibody fusionproteins and fragments thereof for the treatment of B cell lymphomas andleukemias and autoimmune disorders in humans and other mammals. Theantibodies, fusion proteins and fragments thereof can be used alone,conjugated to at least one diagnostic and/or therapeutic agent or incombination with other treatment modalities.

Methods of use of the claimed antibodies may include treatment ofmammalian subjects, such as humans or domestic animals, with one or morehumanized, chimeric or human anti-CD19 antibodies, alone, as an antibodyfusion protein, as a therapeutic conjugate alone or as part of anantibody fusion protein, in combination, or as a multimodal therapy,with other antibodies, other therapeutic agents or immunomodulators oras an immunoconjugate linked to at least one therapeutic agent,therapeutic radionuclide or immunomodulator. These humanized, chimericand human anti-CD19 antibodies can also be used as a diagnostic imagingagent alone, in combination with other diagnostic imaging agents, and/orin conjunction with therapeutic applications. Disease states that may betreated include neoplasias, preferably B cell related lymphomas andleukemias, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia,acute lymphoblastic leukemia, or multiple myeloma. Other disease statesthat may be treated include autoimmune diseases, such as acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosurn, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis ubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, pemphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pernicious anemia, rapidly progressive glomerulonephritis, psoriasis,and fibrosing alveolitis. The skilled artisan will realized that theseare not limiting and any disease state in which CD19 expressing cellsplay a role may potentially be treated with the claimed anti-CD19antibodies, alone or in combination.

Various embodiments concern antibody fusion proteins and fragmentsthereof comprising at least two anti-CD19 MAbs or fragments thereof, orat least one anti-CD19 MAb or fragment thereof and at least one secondMAb or fragment thereof, other than the anti-CD19 MAb or fragmentthereof. Second antibodies of use may include antibodies against other Bcell associated or B cell specific antigens, such as CD20, CD22, CD23,CD80 or HLA-DR. The multispecific and/or fusion proteins can be eithernaked or conjugated to at least one therapeutic and/or diagnostic agent.

The humanized, chimeric and human anti-CD19 MAbs and fragments thereof,and antibody fusion proteins and fragments thereof may be administeredalone, either naked or conjugated to a therapeutic or diagnostic agent,or in combination with another naked antibody, fragment orimmunoconjugate. Also, naked or conjugated anti-CD19 antibodies andfragments thereof, and antibody fusion proteins and fragments thereofmay be administered in combination with at least one therapeutic agentor diagnostic agent that is not conjugated to an anti-CD19 antibody orfragment thereof, or fusion protein or fragment thereof.

Other embodiments relate to DNA sequences encoding a humanized, chimericor human anti-CD19 antibody and fragment thereof, and antibody fusionprotein and fragment thereof. Likewise, a vector and host cellcontaining the DNA sequence is also contemplated. The claimed methodsalso include methods of making the humanized, chimeric and humananti-CD19 antibodies and fragments thereof, and fusion proteins andfragments thereof.

Particular embodiments relate to anti-CD19 MAbs or fragments thereofthat contain specific murine CDRs that have specificity for CD19. TheseMAbs can be humanized, chimeric or human anti-CD19 MAbs. In preferredembodiments, the antibodies may comprise one or more substituted aminoacid residues, such as the substitution of a corresponding murineframework region amino acid residue in a human framework region sequenceof a humanized antibody. Preferred framework region amino acids that arecandidates for substitution include those that are located close oradjacent to one or more CDR amino acid side chains, or that otherwiseaffect the stability and/or expression levels of the encoded protein. Ina most preferred embodiment, the substitution includes replacement of aserine residue with a phenylalanine residue at Kabat residue 91 of theVH sequence of a humanized A19 (hA19) antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 discloses the Vk, the variable light chain, and the VH, thevariable heavy chain, sequences of cA19, a chimeric anti-CD19 antibody.The CDR region sequences are shown in bold and underlined. Thenucleotides are numbered sequentially. Kabat's Ig molecule numbering isused for amino acid residues as shown by the numbering above the aminoacid residues. The amino acid residues numbered by letters are theinsertion residues defined by Kabat numbering scheme. The insertionresidues have the same preceding digits as that of the previous residue.For example, residues 82, 82A, 82B, and 82C in FIG. 1B are indicated as82, A, B, and C, respectively. (A) The light chain variable regionsequences are shown (SEQ ID NO:1 and SEQ ID NO:2). (B) The heavy chainvariable region sequences are shown (SEQ ID NO:3 and SEQ ID NO:4).

FIG. 2 shows the results of cell surface competitive binding assay tocompare the binding specificity of the cA19 antibody with that of otheranti-CD19 antibodies, BU12 and B4. Increasing concentrations of cA19blocked the binding of radiolabeled BU12 to Raji cells in a similarfashion as the unlabeled BU12 and B4, indicating these antibodiesrecognize similar or overlap epitopes of the CD19 molecule.

FIG. 3 compares the amino acid sequences of the variable light chain(Vk) and variable heavy chain (VH) of human antibodies, the chimeric andthe humanized anti-CD19 antibodies. (A) The amino acid sequences of thevariable light chain (Vk) of the human antibody, (REIVk, SEQ ID NO:5), achimeric antibody, (cA19Vk, SEQ ID NO:6), and a humanized antibody,(hA19Vk, SEQ ID NO:7). (B) The amino acid sequences of the variableheavy chain (VH) of the human antibodies, EU (SEQ ID NO:8) and NEWM (FR4only, SEQ ID NO:11), the chimeric antibody, (cA19VH, SEQ ID NO:9) and ahumanized antibody (hA19VH, SEQ ID NO:10).

FIG. 4 discloses the amino acid sequences of the humanized anti-CD19antibody, hA19. (A) DNA and amino acid sequences of the light chain Vgene, (hA19Vk) (SEQ ID NO:12 and SEQ ID NO:13). (B) DNA and amino acidsequences of the heavy chain V gene, hA19VH (SEQ ID NO:14 and SEQ IDNO:15). The nucleotide sequences are shown in lowercase. Numbering of Vkand VH amino acid residues is the same as that in FIG. 1.

FIG. 5 shows the results of cell surface competitive binding assay tocompare the binding specificity and activity of the humanized A19antibody, hA19, with that of cA19. (A) Both unconjugated hA19 (closedtriangles) and cA19 (closed diamonds) blocked the binding of ¹²⁵I-hA19to Raji cells. (B) Both hA19 (closed triangles) and cA19 (closeddiamonds) competed equally well for the binding of ¹²⁵I-cA19 to Rajicells. Increasing concentrations of either cA19 or hA19 blocked thebinding of radiolabeled hA19 or cA19 to Raji cells respectively.

FIG. 6 shows the determination of the Ag-binding affinity (avidity) ofthe anit-CD19 Ab by the direct cell surface binding and Scatchard plotanalysis. Varying concentrations of ¹²⁵I-hA19 (diamonds) or ¹¹²In-cA19(squares) were incubated with Raji cells at 40° C. for 1 h. Total andbound radioactivities were counted and analyzed by Scatchard plot asshown in the inset. hA19 showed virtually same binding affinity as cA19.As shown the apparent dissociation constant values were calculated to be1.1 and 1.2 nM for hA19 and cA19, respectively.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise specified, “a” or “an” as used herein means “one ormore.”

Unless otherwise specified, terms are used in accordance with theirplain and ordinary meaning.

An “antibody,” as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An “antibody fragment” is a portion of an antibody such as F(ab)₂,F(ab′)₂, Fab, Fab′, Fv, sFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-CD19 monoclonal antibody fragmentbinds with an epitope of CD19. Antibody fragments include isolatedfragments consisting of the variable regions, such as “Fv” fragmentsconsisting of the variable regions of the heavy and light chains orrecombinant single chain polypeptide molecules in which light and heavyvariable regions are connected by a peptide linker (“scFv proteins”).

A “naked antibody” is generally an entire antibody which is notconjugated to a therapeutic agent. This is so because the Fc portion ofthe antibody molecule provides effector functions, such as complementfixation and ADCC (antibody dependent cell cytotoxicity), which setmechanisms into action that may result in cell lysis. However, it ispossible that the Fc portion is not required for therapeutic function,with other mechanisms, such as apoptosis, coming into play. “Nakedantibodies” include both polyclonal and monoclonal antibodies, as wellas certain recombinant antibodies, such as chimeric, humanized or humanantibodies.

A “chimeric antibody” is a recombinant protein that contains thevariable domains including the complementarity determining regions(CDRs) of an antibody derived from one species, preferably a rodentantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a rodent antibody, are transferredfrom the heavy and light variable chains of the rodent antibody intohuman heavy and light variable domains. The constant domains of theantibody molecule are derived from those of a human antibody.

A “human antibody” is an antibody that contains human variable andconstant region sequences. For example, human antibodies may be obtainedfrom transgenic mice that have been engineered to produce humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the murine endogenous heavy chain and lightchain loci. The transgenic mice can synthesize human antibodies specificfor human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A fully human antibody also can be constructed bygenetic or chromosomal transfection methods, as well as phage displaytechnology, all of which are known in the art. See for example,McCafferty et al., Nature 348:552-553 (1990) for the production of humanantibodies and fragments thereof in vitro, from immunoglobulin variabledomain gene repertoires from unimmunized donors. In this technique,antibody variable domain genes are cloned in-frame into either a majoror minor coat protein gene of a filamentous bacteriophage, and displayedas functional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. In this way, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats, for their review, see e.g. Johnson and Chiswell, CurrentOpinion in Structural Biology 3:5564-571 (1993). Human antibodies mayalso be generated by in vitro activated B cells. See U.S. Pat. Nos.5,567,610 and 5,229,275, which are incorporated in their entirety byreference.

A “therapeutic agent” is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a sub fragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, enzymes, oligonucleotides, antisense and RNAi oligonucleotides,nucleases, hormones, immunomodulators, chelators, boron compounds,photoactive agents or dyes and radioisotopes.

A “diagnostic agent” is a molecule or atom which may be administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing a disease by locating thecells containing a target antigen. Useful diagnostic agents include, butare not limited to, radioisotopes, dyes, contrast agents,ultrasound-enhancing agents, optical-enhancing agents, fluorescentcompounds or molecules and enhancing agents (e.g. paramagnetic ions) formagnetic resonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRItechnique and the preparation of antibodies conjugated to a MRIenhancing agent and is incorporated in its entirety by reference.Preferably, the diagnostic agents are selected from the group consistingof radioisotopes, enhancing agents for use in magnetic resonanceimaging, ultrasound, and fluorescent compounds. In order to load anantibody component with radioactive metals or paramagnetic ions, it maybe necessary to react it with a reagent having a long tail to which areattached a multiplicity of chelating groups for binding the ions. Such atail can be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chain having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA,porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,polyoximes, and like groups known to be useful for this purpose.Chelates are coupled to the peptides or proteins using standardchemistries. The chelate is normally linked to the antibody by a groupwhich enables formation of a bond to the molecule with minimal loss ofimmunoreactivity and minimal aggregation and/or internal cross-linking.Other, more unusual, methods and reagents for conjugating chelates toantibodies are disclosed in U.S. Pat. No. 4,824,659 to Hawthorne,entitled “Antibody Conjugates”, issued Apr. 25, 1989, the disclosure ofwhich is incorporated herein in its entirety by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I,⁶²Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br,for radio-imaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along with the antibodies of the invention.Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with avariety of metals and radiometals, most particularly with radionuclidesof gallium, yttrium and copper, respectively. Such metal-chelatecomplexes can be made very stable by tailoring the ring size to themetal of interest. Other ring-type chelates such as macro cyclicpolyethers, which are of interest for stably binding nuclides, such as²²³Ra for RAIT are encompassed.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic or diagnostic agent.

An “expression vector” is a nucleic acid molecule, preferably adouble-stranded DNA molecule, comprising a gene that is expressed in ahost cell. Typically, gene expression is placed under the control ofcertain regulatory elements, including constitutive or induciblepromoters, tissue-specific regulatory elements and enhancers. Such agene is said to be “operably linked to” the regulatory elements.

A “recombinant host cell” may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as a transgenicanimal, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells of thehost animal. Suitable mammalian host cells include myeloma cells, suchas SP2/0 cells, and NS0 cells, as well as Chinese Hamster Ovary (CH0)cells, hybridoma cell lines and other mammalian host cells useful forexpressing antibodies. Also useful to express MAbs and other fusionproteins, is a human cell line, PER.C6 disclosed in WO 0063403 A2. Mostpreferred host cells are cells that have been engineered to contain aBcl-EEE gene or other apoptosis inhibitor, that have been pre-adapted togrow and be transfected in serum-free or low-serum media. Examples ofsuch host cells are disclosed in U.S. patent application Ser. Nos.11/187,863, filed Jul. 25, 2005 and 11/487,215, filed Jul. 14, 2006, thetext of each of which is incorporated herein by reference in itsentirety. Special transgenic animals with a modified immune system areparticularly useful for making fully human antibodies.

As used herein, the term “antibody fusion protein” refers to arecombinantly produced antigen-binding molecule comprising one or moreof the same or different single-chain antibody or antibody fragmentsegments with the same or different specificities. Antibody fusionproteins may comprise an antibody or fragment thereof attached toanother protein or peptide, such as a therapeutic agent, toxin,cytokine, hormone or other protein or peptide. In other embodiments,antibody fusion proteins may comprise at least a first and secondantibody or antibody fragment. Where fusion proteins comprise two ormore antibodies or fragments, the valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. Specificity indicates how many antigens or epitopes anantibody fusion protein is able to bind; i.e., monospecific, bispecific,bispecific, multispecific. Using these definitions, a natural antibody,e.g., an IgG, is bivalent because it has two binding arms but ismonospecific because it binds to one epitope.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope.Another specificity could be to a different antigen on the same celltype, such as CD20, CD19, CD20, CD21, CD23, CD46, CD80, HLA-DR, CD74,and CD22 on B-cells. Multispecific, multivalent antibodies areconstructs that have more than one binding site, and the binding sitesare of different specificity. For example, a diabody, where one bindingsite reacts with one antigen and the other with another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-,and mast-cell antigen or epitope and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering.

Domestic animals include large animals such as horses, cattle, sheep,goats, llamas, alpacas, and pigs, as well as companion animals. In apreferred embodiment, the domestic animal is a horse. Companion animalsinclude animals kept as pets. These are primarily dogs and cats,although small rodents, such as guinea pigs, hamsters, rats, andferrets, are also included, as are subhuman primates such as monkeys. Ina preferred embodiment the companion animal is a dog or a cat.

Overview

As discussed above, anti-CD19 antibodies that are unconjugated orlabeled with a therapeutic radionuclide, have failed to provide highrates of objective and lasting responses in patients with intermediateor aggressive forms of B-cell lymphoma. The present invention provideshumanized, chimeric or human anti-CD19 antibodies and antibody fusionproteins useful for treatment of mammalian subjects, humans and domesticanimals, alone, as a conjugate or administered in combination with othertherapeutic agents, including other naked antibodies and antibodytherapeutic conjugates.

The anti-CD19 MAbs preferably contain specific murine CDRs from one ormore murine or chimeric anti-CD19 MAbs that have specificity for theCD19 antigen. The anti-CD19 MAbs are humanized, chimeric or human MAbs.The CDRs of the light chain variable region of the anti-CD19 MAbpreferably comprises CDR1 comprising amino acids KASQSVDYDGDSYLN (SEQ IDNO:16); CDR2 comprising amino acids DASNLVS (SEQ ID NO:17); and CDR3comprising amino acids QQSTEDPWT (SEQ ID NO:18); and the heavy chainvariable region CDR1 comprising amino acids SYWMN (SEQ ID NO:19); CDR2comprising amino acids QIWPGDGDTNYNGKFKG (SEQ ID NO:20) and CDR3comprising amino acids RETTTVGRYYYAMDY (SEQ ID NO:21).

In a preferred embodiment, the humanized anti-CD19 MAb or fragmentthereof comprises the CDRs of a murine anti-CD19 MAb and the framework(FR) regions of the light and heavy chain variable regions of a humanantibody and the light and heavy chain constant regions of a humanantibody, while retaining substantially the B-cell, and B-cell lymphomaand leukemia cell targeting of the parent murine anti-CD19 MAb, andwherein the CDRs of the light chain variable region of the anti-CD19 MAbcomprise CDR1 comprising amino acids KASQSVDYDGDSYLN (SEQ ID NO:16);CDR2 comprising amino acids DASNLVS (SEQ ID NO:17); and CDR3 comprisingamino acids QQSTEDPWT (SEQ ID NO:18); and the CDRs of the heavy chainvariable region of the anti-CD19 MAb comprise CDR1 comprising aminoacids SYWMN (SEQ ID NO:19); CDR2 comprising amino acidsQIWPGDGDTNYNGKFKG (SEQ ID NO:20) and CDR3 comprising amino acidsRETTTVGRYYYAMDY (SEQ ID NO:21). The humanized anti-CD19 MAb or fragmentthereof may further contain in the FRs of the light and heavy chainvariable regions of the antibody at least one amino acid from thecorresponding FRs of the murine MAb. Specifically, the humanizedanti-CD19 MAb or fragment thereof may contain at least one amino acidresidue selected from residues 5, 27, 28, 40, 48, 91, 94, 107 and 108 ofthe murine heavy chain variable region of FIG. 4A and at least one aminoacid residue selected from residues 4, 39, 58, 60, 87, 100, and 107 ofthe murine light chain variable region FIG. 4B. In a more preferredembodiment, the humanized A19 antibody (hA19) contains each of thesubstituted murine FR amino acid residues listed above. One or more ofthe murine amino acid sequences can be maintained in the human FRregions of the light and heavy variable chains if necessary to maintainproper binding or to enhance binding to the CD19 antigen. Morepreferably the humanized anti-CD19 MAb or fragment thereof comprises thehA19Vk (SEQ ID NO:7) of FIG. 3A and the hA19VH (SEQ ID NO:10) of FIG.3B. Most preferably, the humanized anti-CD19 MAb comprises an additionalFR substitution of a serine residue with a phenylalanine residue atKabat residue 91 of the hA19VH (SEQ ID NO:10) sequence.

The preferred chimeric anti-CD19 (cA19) MAb or fragment thereofcomprises the CDRs of a murine anti-CD19 MAb and the FR regions of thelight and heavy chain variable regions of the murine anti-CD19 MAb,i.e., the Fvs of the parental murine MAb, and the light and heavy chainconstant regions of a human antibody, wherein the chimeric anti-CD19 MAbor fragment thereof retains substantially the B-cell, and B-celllymphoma and leukemia cell targeting of the murine anti-CD19 MAb,wherein the CDRs of the light chain variable region of the anti-CD19 MAbcomprises CDR1 comprising amino acids KASQSVDYDGDSYLN (SEQ ID NO:16);CDR2 comprising amino acids DASNLVS (SEQ ID NO:17); and CDR3 comprisingamino acids QQSTEDPWT (SEQ ID NO:18); and the CDRs of the heavy chainvariable region of the anti-CD I 9 MAb comprises CDR1 comprising aminoacids SYWMN (SEQ ID NO:19); CDR2 comprising amino acidsQ1WPGDGDTNYNGKFKG and CDR3 comprising amino acids RETTTVGRYYYAMDY (SEQID NO:21). More preferably the chimeric anti-CD19 MAb or fragmentthereof comprises the light and heavy chain variable region sequences ofthe chimeric anti-CD19 MAb shown in FIGS. 1A and 1B, respectively,designated cA19Vk (SEQ ID NO:6) and cA19VH (SEQ ID NO:9).

Various embodiments also encompass a human anti-CD19 MAb or fragmentthereof comprising the light and heavy chain variable and constantregions of a human antibody, wherein said human anti-CD19 MAb retainssubstantially the B-cell, and B-cell lymphoma and leukemia celltargeting and cell binding characteristics of a murine anti-CD19 MAb,wherein the CDRs of the light chain variable region of the humananti-CD19 MAb comprises the same CDRs (SEQ ID NOs:16-21) as set forthabove for the chimeric and humanized anti-CD19 MAbs and as shown inFIGS. 1A and 1B, and 3A and 3B, respectively.

Certain embodiments are also intended to encompass antibody fusionproteins or fragments thereof comprising at least two anti-CD19 MAbs orfragments thereof, as described above. The antibody fusion protein orfragment thereof is also intended to encompass an antibody fusionprotein or fragment thereof comprising at least one first anti-CD19 MAbor fragment thereof as described above and at least one second MAb orfragment thereof, other than the anti-CD19 MAb or fragment describedabove. More preferably this second MAb is a MAb reactive with CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia,HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, P1GF, ED-Bfibronectin, an oncogene, an oncogene product, CD66a-d, necrosisantigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5)or a combination thereof, and even an anti-CD19 MAb that is directed toa different epitope than the anti-CD19 MAb described herein. Theantibody fusion proteins may be composed of one anti-CD19 MAb and one ormore of the second MAbs to provide specificity to different antigens,and are described in more detail below.

The humanized, chimeric and human anti-CD19 antibody may possessenhanced affinity binding with the epitope, as well as antitumor andanti-B-cell activity, as a result of amino acid mutation andmanipulation of the sequences in the variable region to obtain asuperior therapeutic agent for the treatment of B-cell disorders,including B-cell lymphomas and leukemias and autoimmune diseases.Modification to the binding specificity, affinity or avidity of anantibody is known and described in WO 98/44001, as affinity maturation,and this application summarizes methods of modification and isincorporated in its entirety by reference.

It may also be desirable to modify the antibodies to improve effectorfunction, e.g., so as to enhance antigen-dependent cell-mediatedcytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) ofthe antagonist. One or more amino acid substitutions or the introductionof cysteine in the Fc region may be made, thereby improvinginternalization capability and/or increased complement-mediated cellkilling and ADCC. See Caron et al., J. Ex. Med. 176:1191-1195 (1991) andShopes, Brit. J. Immunol. 148:2918-2022 (1992), incorporated herein byreference in their entirety. An antibody fusion protein may be preparedthat has dual Fc regions with both enhanced complement lysis and ADCCcapabilities.

Certain embodiments are also directed to DNA sequences comprising anucleic acid encoding a MAb or fragment thereof selected from the groupconsisting of: (a) an anti-CD19 MAb or fragment thereof as describedherein, (b) an antibody fusion protein or fragment thereof comprising atleast two of the anti-CD19 MAbs or fragments thereof, (c) an antibodyfusion protein or fragment thereof comprising at least one first MAb orfragment thereof comprising the anti-CD19 MAb or fragment thereof asdescribed herein and at least one second MAb or fragment thereof, otherthan the antiCD19 MAb or fragment thereof, and (d) an antibody fusionprotein or fragment thereof comprising at least one first MAb orfragment thereof comprising the anti-CD19 MAb or fragment thereof and atleast one second MAb or fragment thereof, wherein the second MAb is aMAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, MUC1, 1a, HM1.24, HLA-DR, tenescin, ED-B fibronectin,IL-6, VEGF, PIGF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) or a combinationthereof.

Also encompassed are expression vectors comprising the DNA sequences.These vectors contain the light and heavy chain constant regions and thehinge region of the human immunoglobulin, in the case of vectors for usein preparing the humanized, chimeric and human anti-CD19 MAbs orantibody fusion proteins thereof or fragments thereof. These vectorsadditionally contain, where required, promoters that express the MAbs inthe selected host cell, immunoglobulin enhancers and signal or leadersequences. Vectors that are particularly useful in the present inventionare pdHL2 or GS, particularly when used to express a chimeric, humanizedor human antibody, such as IgGs, where the vector codes for the heavyand light chain constant regions and hinge region of IgG1. Morepreferably, the light and heavy chain constant regions and hinge regionare from a human ED myeloma immunoglobulin, where optionally at leastone of the amino acids in the allotype positions is changed to thatfound in a different IgG1 allotype, and wherein optionally amino acid253 of the heavy chain of EU based on the EU number system may bereplaced with alanine. See Edelman et al., Proc. Natl. Acad. Sci. USA63: 78-85 (1969), incorporated herein in its entirety by reference.

Host cells containing the DNA sequences encoding the anti-CD19 MAbs orfragments thereof or antibody fusion proteins or fragments thereof orhost cells containing the vectors that contain these DNA sequences areencompassed by the present invention. Particularly useful host cells aremammalian cells, more specifically lymphocytic cells, such as myelomacells, discussed in more detail below.

Also encompassed are methods of expressing the anti-CD19 MAb or fragmentthereof or antibody fusion protein or fragment thereof comprising: (a)transfecting a mammalian cell with a DNA sequence encoding the anti-CD19MAbs or fragments thereof or antibody fusion proteins or fragmentsthereof, and (b) culturing the cell transfected with the DNA sequencethat secretes the anti-CD19 or fragment thereof or antibody fusionprotein or fragment thereof. Known techniques may be used that include aselectable marker on the vector so that host cells that express the MAbsand the marker can be easily selected.

The present invention particularly encompasses B-lymphoma cell, leukemiacell and/or autoimmune cell targeting diagnostic or therapeuticconjugates comprising an antibody component comprising an anti-CD19 MAbor fragment thereof or an antibody fusion protein or fragment thereofthat binds to the target cell that is conjugated or otherwise attachedto at least one diagnostic or at least one therapeutic agent.

The diagnostic conjugate comprises an antibody component comprising ananti-CD19 MAb or fragment thereof or an antibody fusion protein orfragment thereof, wherein the diagnostic agent comprises at least onephotoactive diagnostic agent, more preferably wherein the label is aradioactive label with an energy between 60 and 4,000 keV or anon-radioactive label. The radioactive label is preferably a gamma-,beta- or positron-emitting isotope and is selected from the groupconsisting of ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶²Cu, ⁶⁴Cu,¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br andcombinations thereof.

The diagnostic conjugate may utilize a diagnostic agent, such as acontrast agent, for example, such as manganese, iron or gadolinium, orincluding an ultrasound-enhancing agent. In one embodiment, theultrasound-enhancing agent is a liposome that comprises a chimerized orhumanized anti-CD19 antibody or fragment thereof. Also preferred, theultrasound enhancing agent is a liposome that is gas filled. Similarly,a bispecific antibody can be conjugated to a contrast agent. Forexample, the bispecific antibody may comprise more than one imageenhancing agent for use in ultrasound imaging. The ultrasound enhancingagent can be a liposome, and preferably, the liposome comprises abivalent DTP A peptide covalently attached to the outside surface of theliposome. Also preferred, the liposome is gas filled.

The therapeutic conjugate comprises an antibody component, such as anantibody fusion protein or fragment thereof, wherein each of said MAbsor fragments thereof are bound to at least one therapeutic agent. Thetherapeutic conjugate preferably is selected from the group consistingof a radioactive label, an immunomodulator, a hormone, an enzyme, anoligonucleotide, a photoactive therapeutic agent, a cytotoxic agent,which may be a drug or a toxin, and a combination thereof. Useful drugsinclude those drugs that possess a pharmaceutical property selected fromthe group consisting of antimitotic, alkylating, antimetabolite,antibiotic, alkaloid, antiangiogenic, apoptotic agents and combinationsthereof, as well as antisense oligonucleotides and RNA molecules, suchas short double stranded RNA molecules that activate the RNAinterference pathway. More specifically, the drugs may be selected fromthe group consisting of nitrogen mustards, ethylenimine derivatives,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, thalidomide and its derivatives, antagonists, endostatin,taxols, camptothecins, anthracyclines, taxanes, and their analogs, and acombination thereof. The toxins may be selected from the groupconsisting of ricin, abrin, alpha toxin, saporin, onconase, i.e.,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.

Other therapeutic agents suitable for use include anti-angiogenic agents(or angiogenesis inhibitors). These agents are suitable for use incombination therapy or for conjugating antibodies to, for example,angiostatin, endostatin, vasculostatin, canstatin and maspin, as well asthe use of antibodies against angiogenesis factors, such as vascularendothelium growth factor (VEGF), placental growth factor (P1GF), ED-Bfibronectin, and against other vascular growth factors. Single anddouble stranded oligonucleotides are a new class of therapeutic agents,and include, for example, antisense oligonucleotides, such as antisensebc1-2, and molecules, such as double stranded RNA molecules, thatactivate the RNA interference pathway and cause highly specificinhibition of gene expression, such as inhibition of be 1-2. Inhibitionof be 1-2 (and related bc1 family molecules) in a cell inhibits theanti-apoptotic activity of bc1-2 and promotes apoptosis of the cell. SeeZangemeister-Wittke, Ann N Y Acad. Sci. 1002:90-4 (2003).

Useful therapeutic conjugates are immunomodulators selected from thegroup consisting of a cytokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), erythropoietin, thrombopoietin and a combinationthereof. Specifically useful are lymphotoxins, such as tumor necrosisfactor (TNF), hematopoietic factors, such as interleukin (IL), colonystimulating factor, such as granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF),interferon, such as interferons -alpha, -beta or -gamma, and stem cellgrowth factor, such as “S1 factor”. More specifically, immunomodulators,such as IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon,TNF-alpha or -beta or a combination thereof may be of use.

Particularly useful therapeutic conjugates include one or moreradioactive labels that have an energy between 60 and 700 keV. Suchradioactive labels may be selected from the group consisting of ²²⁵Ac,⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹³¹I, ¹²⁵I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ³²P, ⁶⁴Cu, ⁶⁷Cu,²¹²Bi, ²¹³Bi, ²¹¹At and combinations thereof. Other useful therapeuticconjugates are photoactive therapeutic agents, such as a chromogen ordye.

The claimed methods encompass methods of treating a B-cell disease in asubject, such as a mammal, including humans, domestic or companion pets,such as dogs and cats. B cell diseases that can be treated by themethods include any disease which involves unwanted or undesirable Bcell growth or activity, and includes malignancies such as lymphoma orleukemia or an autoimmune disease. The methods involve administering tothe subject a therapeutically effective amount of an anti-CD19 MAb or afragment thereof, formulated in a pharmaceutically acceptable vehicle.This therapy may utilize a “naked antibody” that does not have atherapeutic agent bound to it. The administration of the “nakedanti-CD19 antibody” can be supplemented by administering to the subjectconcurrently or sequentially a therapeutically effective amount ofanother therapeutic agent, such as a second “naked antibody” that bindsto or is reactive with another antigen on the surface of the target cellor that has other functions, such as effector functions in the Fcportion of the MAb, that is therapeutic and which is discussed herein.Preferred additional MAbs are at least one humanized, chimeric, human ormurine (in the case of non-human animals) MAb selected from the groupconsisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20,CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD45, CD46,CD52, CD54, CD74, CD80, CD126, CD138, B7, HM1.24, HLA-DR, anangiogenesis factor, tenascin, VEGF, P1GF, ED-B fibronectin, anoncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2,MUC-1, TO1, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) formulated in apharmaceutically acceptable vehicle. In other embodiments, the anti-CD19antibody may be conjugated to one or more therapeutic or diagnosticagents.

Both the naked anti-CD19 therapy alone or in combination with othernaked MAbs as discussed above can be further supplemented with theadministration, either concurrently or sequentially, of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle. As discussed hereinthe therapeutic agent may comprise a cytotoxic agent, a radioactivelabel, an immunomodulator, a hormone, an oligonucleotide (such as anantisense or RNAi oligonucleotide), an enzyme, a photoactive therapeuticagent or a combination thereof, formulated in a pharmaceuticallyacceptable vehicle.

In another therapeutic method, both the naked anti-CD19 therapy alone orin combination with other naked MAbs, as discussed above, can be furthersupplemented with the administration, either concurrently orsequentially, of a therapeutically effective amount of at least onetherapeutic conjugate, described herein and formulated in apharmaceutically acceptable vehicle. The antibody component of thetherapeutic conjugate comprises at least one humanized, chimeric, humanor murine (for non-human subjects) MAb selected from the groupconsisting of a MAb reactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54,CD74, CD80, CD126, CD138, B7, HM1.24, HLA-DR, an angiogenesis factor,tenascin, VEGF, P1GF, ED-B fibronectin, an oncogene, an oncogeneproduct, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, MUC-1,TRAIL-R1 (DR4) and TRAIL-R2 (DR5), formulated in a pharmaceuticallyacceptable vehicle. As discussed herein the therapeutic agent maycomprise a cytotoxic agent, a radioactive label, an immunomodulator, ahormone, a photoactive therapeutic agent or a combination thereof,formulated in a pharmaceutically acceptable vehicle.

Other embodiments concern methods of treating a B-cell lymphoma orleukemia or an autoimmune disease in a subject comprising administeringto a subject a therapeutically effective amount of an antibody fusionprotein or fragment thereof comprising at least two anti-CD19 MAbs orfragments thereof or comprising at least one anti-CD19 MAb or fragmentthereof and at least one additional MAb, preferably selected from thegroup consisting of MAbs reactive with CD4, CD5, CD8, CD14, CD15, CD19,CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52,CD54, CD74, CD80, CD126, CD138, B7, HM1.24, HLA-DR, tenascin, VEGF,P1GF, ED-B fibronectin, MUC-1, an oncogene, an oncogene product,CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4)and TRAIL-R2 (DR5) formulated in a pharmaceutically acceptable vehicle.

In other methods based on pretargeting techniques, a multispecificantibody or fusion protein may comprise an anti-CD19 antibody asdescribed herein, attached to at least one other antibody or fragmentthat binds to a hapten, such as an HSG hapten. The hapten may beincorporated into a targetable conjugate that comprises one or moretherapeutic and/or diagnostic agents. The multispecific antibody orfusion protein may be administered to a subject and allowed to bind to atarget antigen, such as CD19, expressed on a target cell, such as a Bcell. After allowing a sufficient amount of time for non-boundcirculating antibody to be removed from circulation, the targetableconjugate may be administered, which then binds to the multispecificantibody or fusion protein localized to the target cell or tissue. Suchpretargeting methods improve the therapeutic index by preferentialdelivery of therapeutic agent to the target cell or tissue compared tonormal cells or tissues. Methods of pretargeting are well known in theart (see, e.g., U.S. Pat. Nos. 6,361,774; 6,962,702; 7,074,403;7,201,890; 7,230,084; 7,230,085 and 7,429,381, each incorporated hereinby reference in its entirety.)

The therapeutic methods can further be supplemented with theadministration to the subject concurrently or sequentially of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle, wherein thetherapeutic agent is preferably a cytotoxic agent, a radioactive label,an immunomodulator, a hormone, a photoactive therapeutic agent or acombination thereof, formulated in a pharmaceutically acceptablevehicle.

Further, the antibody fusion proteins can be administered to a subjectconcurrently or sequentially with a therapeutically effective amount ofa therapeutic conjugate comprising at least one MAb bound to at leastone therapeutic agent, wherein said MAb component of the conjugatepreferably comprises at least one humanized, chimeric, human or murine(for non-human subjects) MAb selected from the group consisting of a MAbreactive with CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, CD138, B7, MUC1, Ia, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-Bfibronectin, an oncogene, an oncogene product, CD66a-d, necrosisantigens, Ii, IL-2, IL-6, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2(DR5) formulated in a pharmaceutically acceptable vehicle. The antibodyfusion protein itself can be conjugated to a therapeutic agent and thusprovides a vehicle to attach more than one therapeutic agent to anantibody component and these therapeutic agents can be a combination ofdifferent recited agents or combinations of the same agents, such as twodifferent therapeutic radioactive labels.

Also encompassed are methods of diagnosing or detecting a B-celllymphoma or leukemia or autoimmune disease in a subject comprisingadministering to the subject, such as a mammal, including humans anddomestic and companion pets, such as dogs, cats, rabbits, guinea pigs, adiagnostic conjugate comprising an anti-CD19 MAb or fragment thereof oran antibody fusion protein or fragment thereof that binds to thelymphoma, leukemia or autoimmune cell, wherein the anti-CD19 MAb orfragment thereof or antibody fusion protein or fragment thereof is boundto at least one diagnostic agent, formulated in a pharmaceuticallyacceptable vehicle. Useful diagnostic agents are described herein.

Antibody Preparation

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen wherein the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into human variable domains attached tohuman constant region sequences, and then, substituting selected humanresidues in the framework regions with their murine counterparts.Preferred FR residues for substitution include those FR residues thatare located near or touching the CDR residues, as well as residues thataffect the stability and/or expression levels of the antibody. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

General techniques for cloning murine immunoglobulin variable domainsare disclosed, for example, by the publication of Orlandi et al., Proc.Natl. Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et at.,Hybridoma 13:469 (1994), produced an LL2 chimera by combining DNAsequences encoding the Vk and VH domains of LL2 monoclonal antibody, ananti-CD22 antibody, with respective human and IgG1 constant regiondomains. This publication also provides the nucleotide sequences of theLL2 light and heavy chain variable regions, Vk and VH, respectively.Techniques for producing humanized MAbs are described, for example, byJones et al., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323(1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc.Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J. Immun. 150: 2844 (1993), each of whichis incorporated herein by reference.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric MAbwith one or more different human FR. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988).

Another method for producing the antibodies is by production in the milkof transgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63:141-147, 1998; U.S. Pat. No. 5,827,690, both of which are incorporatedin their entirety by reference. Two DNA constructs are prepared whichcontain, respectively, DNA segments encoding paired immunoglobulin heavyand light chains. The DNA segments are cloned into expression vectorswhich contain a promoter sequence that is preferentially expressed inmammary epithelial cells. Examples include, but are not limited to,promoters from rabbit, cow and sheep casein genes, the cow-lactoglobulingene, the sheep-lactoglobulin gene and the mouse whey acid protein gene.Preferably, the inserted fragment is flanked on its 3′ side by cognategenomic sequences from a mammary-specific gene. This provides apolyadenylation site and transcript-stabilizing sequences. Theexpression cassettes are coinjected into the pronuclei of fertilized,mammalian eggs, which are then implanted into the uterus of a recipientfemale and allowed to gestate. After birth, the progeny are screened forthe presence of both transgenes by Southern analysis. In order for theantibody to be present, both heavy and light chain genes must beexpressed concurrently in the same cell. Milk from transgenic females isanalyzed for the presence and functionality of the antibody or antibodyfragment using standard immunological methods known in the art. Theantibody can be purified from the milk using standard methods known inthe art.

A fully human antibody, i.e., human anti-CD19 MAbs or other humanantibodies, such as anti-CD22, anti-CD23, anti-CD20, anti-CD74 oranti-CD21 MAbs, can be obtained from a transgenic non-human animal. See,e.g., Mendez et al., Nature Genetics, 15: 146-156 (1997); U.S. Pat. No.5,633,425, which are incorporated in their entirety by reference. Forexample, a human antibody can be recovered from a transgenic mousepossessing human immunoglobulin loci. The mouse humoral immune system ishumanized by inactivating the endogenous immunoglobulin genes andintroducing human immunoglobulin loci. The human immunoglobulin loci areexceedingly complex and comprise a large number of discrete segmentswhich together occupy almost 0.2% of the human genome. To ensure thattransgenic mice are capable of producing adequate repertoires ofantibodies, large portions of human heavy- and light-chain loci must beintroduced into the mouse genome. This is accomplished in a stepwiseprocess beginning with the formation of yeast artificial chromosomes(YACs) containing either human heavy or light-chain immunoglobulin lociin germline configuration. Since each insert is approximately 1 Mb insize, YAC construction requires homologous recombination of overlappingfragments of the immunoglobulin loci. The two YACs, one containing theheavy-chain loci and one containing the light-chain loci, are introducedseparately into mice via fusion of YAC-containing yeast spheroblastswith mouse embryonic stem cells. Embryonic stem cell clones are thenmicroinjected into mouse blastocysts. Resulting chimeric males arescreened for their ability to transmit the Y A C through their germlineand are bred with mice deficient in murine antibody production. Breedingthe two transgenic strains, one containing the human heavy-chain lociand the other containing the human light-chain loci, creates progenywhich produce human antibodies in response to immunization.

Further recent methods for producing bispecific MAbs include engineeredrecombinant MAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering. See, for example, Alt et al.,FEBS Lett. 454:90-4 (1999), which is incorporated herein by reference inits entirety. In one form, the bispecific fusion protein consists of,for example, a scFv with a single binding site for one antigen and a Fabfragment with a single binding site for a second antigen. In anotherform, the bispecific fusion protein consists of, for example, an IgGwith two binding sites for one antigen and two scFv with two bindingsites for a second antigen.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.For example a fusion protein comprising a Fab fragment derived from ahumanized monoclonal anti-CD19 antibody and a scFv derived from a murineanti-diDTPA can be produced. A flexible linker, such as GGGS connectsthe scFv to the constant region of the heavy chain of the anti-CD19antibody. Alternatively, the scFv can be connected to the constantregion of the light chain of another humanized antibody. Appropriatelinker sequences necessary for the in-frame connection of the heavychain Fd to the scFv are introduced into the VL and Vk domains throughPCR reactions. The DNA fragment encoding the scFv is then ligated into astaging vector containing a DNA sequence encoding the CH1 domain. Theresulting scFv-CH1 construct is excised and ligated into a vectorcontaining a DNA sequence encoding the VH region of an anti-CD19antibody. The resulting vector can be used to transfect an appropriatehost cell, such as a mammalian cell for the expression of the bispecificfusion protein.

More recently, a novel technique known as dock-and-lock (DNL) has beendeveloped to provide for the highly efficient formation of constructscomprising virtually any combination of peptide or protein effectors(see, e.g., U.S. Patent Application Publ. Nos. 20060228357; 20060228300;20070086942; 20070140966 and 20070264265, each incorporated herein byreference in its entirety). In various permutations, the constructs arenot limited to protein or peptide effectors, but may comprise othertypes of effector agents that may be attached to proteins or peptides,such as chemotherapeutic agents. The technique utilizes complementaryprotein binding domains, referred to as anchoring domains anddimerization and docking domains, which bind to each other and allow theassembly of complex structures, ranging from dimers, trimers, tetramers,quintamers and hexamers. These form stable complexes in high yieldwithout requirement for extensive purification. The DNL technique allowsthe assembly of monospecific, bispecific or multispecific antibodies,either as naked antibody moieties or in combination with a wide range ofother effector molecules such as immunomodulators, enzymes,chemotherapeutic agents, chemokines, cytokines, diagnostic agents,therapeutic agents, radionuclides, imaging agents, anti-angiogenicagents, growth factors, oligonucleotides, hormones, peptides, toxins,pro-apoptotic agents, or a combination thereof. Any of the techniquesknown in the art for making bispecific or multispecific antibodies maybe utilized in the practice of the presently claimed methods.

Known Antibodies

In certain embodiments, for example those involving bispecific ormultispecific antibodies incorporating an anti-CD19 antibody and anotherantibody against a different antigenic target, it may be preferred toutilize a commercially available or publicly known antibody, rather thanmaking one de novo. Antibodies of use may be commercially obtained froma wide variety of known sources. For example, a variety of antibodysecreting hybridoma lines are available from the American Type CultureCollection (ATCC, Manassas, Va.). A large number of antibodies againstvarious disease targets, including but not limited to tumor-associatedantigens, have been deposited at the ATCC and/or have published variableregion sequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, incorporated herein by reference with respect to antibodyvariable region and/or CDR sequences and/or ATCC Accession Numbers forantibody-producing hybridoma cell lines. These are exemplary only and awide variety of other antibodies and their hybridomas are known in theart. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art.

Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.Other antibody fragments include, but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab′expression libraries can be constructed (Huse et al., 1989, Science,246:1274-1281) to allow rapid and easy identification of monoclonal Fab′fragments with the desired specificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TIBTECH, Vol 9: 132-137(1991), each incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fabmonovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, incorporatedherein in their entireties by reference. Also, see Nisonoff et al., ArchBiochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959),Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (AcademicPress 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the same antigen that isrecognized by the intact antibody.

Multispecific and Multivalent Antibodies

The anti-CD19 antibodies, as well as other antibodies with differentspecificities for use in combination therapy, can also be made asmultispecific antibodies, comprising at least one binding site to a CD19epitope or antigen and at least one binding site to another epitope onCD19 or another antigen, or multivalent antibodies, comprising multiplebinding sites to the same epitope or antigen.

A bispecific antibody or antibody fragment may have at least one bindingregion that specifically binds a targeted cell marker and at least oneother binding region that specifically binds a targetable conjugate. Thetargetable conjugate comprises a carrier portion which comprises orbears at least one epitope recognized by at least one binding region ofthe bispecific antibody or antibody fragment. A variety of recombinantmethods can be used to produce bispecific antibodies and antibodyfragments as described above.

An anti-CD19 multivalent antibody is also contemplated. This multivalenttarget binding protein is constructed by association of a first and asecond polypeptide. The first polypeptide comprises a first single chainFv molecule covalently linked to a first immunoglobulin-like domainwhich preferably is an immunoglobulin light chain variable regiondomain. The second polypeptide comprises a second single chain Fvmolecule covalently linked to a second immunoglobulin-like domain whichpreferably is an immunoglobulin heavy chain variable region domain. Eachof the first and second single chain Fv molecules forms a target bindingsite, and the first and second immunoglobulin-like domains associate toform a third target binding site.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment is an anti-CD19 bispecific, trivalent targetingprotein comprising two heterologous polypeptide chains associatednoncovalently to form three binding sites, two of which have affinityfor one target and a third which has affinity for a hapten that can bemade and attached to a carrier for a diagnostic and/or therapeuticagent. Preferably, the binding protein has two CD19 binding sites andone CD22 binding site. The bispecific, trivalent targeting agents havetwo different scFvs, one scFv contains two VH domains from one antibodyconnected by a short linker to the VL domain of another antibody and thesecond scFv contains two VL domains from the first antibody connected bya short linker to the VH domain of the other antibody. The methods forgenerating multivalent, multispecific agents from VH and VL domainsprovide that individual chains synthesized from a DNA plasmid in a hostorganism are composed entirely of VH domains (the VH-chain) or entirelyof VL domains (the VL-chain) in such a way that any agent ofmultivalency and multispecificity can be produced by non-covalentassociation of one VH-chain with one VL-chain. For example, forming atrivalent, trispecific agent, the VH-chain will consist of the aminoacid sequences of three VH domains, each from an antibody of differentspecificity, joined by peptide linkers of variable lengths, and theVL-chain will consist of complementary VL domains, joined by peptidelinkers similar to those used for the VH-chain. Since the VH and VLdomains of antibodies associate in an anti-parallel fashion, thepreferred method in this invention has the VL domains in the VL-chainarranged in the reverse order of the VH domains in the VH-chain.

Diabodies, Triabodies and Tetrabodies

The anti-CD19 antibodies can also be used to prepare functionalbispecific single-chain antibodies (bsAb), also called diabodies, andcan be produced in mammalian cells using recombinant methods. See, e.g.,Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995, incorporatedherein by reference. For example, bsAb are produced by joining twosingle-chain Fv fragments via a glycine-serine linker using recombinantmethods. The V light-chain (VL) and V heavy-chain (VH) domains of twoantibodies of interest are isolated using standard PCR methods. The VLand VH cDNAs obtained from each hybridoma are then joined to form asingle-chain fragment in a two-step fusion PCR. The first PCR stepintroduces the (Gly4-Ser1)₃ linker, and the second step joins the VL andVH amplicons. Each single chain molecule is then cloned into a bacterialexpression vector. Following amplification, one of the single-chainmolecules is excised and sub-cloned into the other vector, containingthe second single-chain molecule of interest. The resulting bsAbfragment is subcloned into a eukaryotic expression vector. Functionalprotein expression can be obtained by transfecting the vector into CHOcells, Sp2/0 cells or Sp-EEE cells. Bispecific fusion proteins areprepared in a similar manner.

For example, a humanized, chimeric or human anti-CD19 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the VH polypeptide of the humanizedanti-CD19 MAb connected to the VK polypeptide of the humanized anti-CD19MAb by a five amino acid residue linker are utilized. Each chain formsone half of the humanized anti-CD19 diabody. In the case of triabodies,the three chains comprising VH polypeptide of the humanized anti-CD19MAb connected to the VK polypeptide of the humanized anti-CD19 MAb by nolinker are utilized. Each chain forms one third of the hCD19 triabody.

The ultimate use of the bispecific diabodies described herein is forpretargeting CD19 positive tumors for subsequent specific delivery ofdiagnostic or therapeutic agents. These diabodies bind selectively totargeted antigens allowing for increased affinity and a longer residencetime at the desired location. Moreover, non-antigen bound diabodies arecleared from the body quickly and exposure of normal tissues isminimized. Bispecific antibody point mutations for enhancing the rate ofclearance can be found in U.S. Provisional Application No. 60/361,037,which is incorporated herein by reference in its entirety. Bispecificdiabodies for affinity enhancement are disclosed in U.S. applicationSer. No. 10/270,071, Ser. No. 10/270,073 and Ser. No. 10/328,190, whichare incorporated herein by reference in their entirety.

The diagnostic and therapeutic agents can include isotopes, drugs,toxins, cytokines, hormones, enzymes, oligonucleotides, growth factors,conjugates, radionuclides, and metals. For example, gadolinium metal isused for magnetic resonance imaging (MRI). Examples of radio nuclidesare ²²⁵Ac, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In, ¹³¹I, ¹²⁵I, ¹²³I, ^(99m)Tc,^(94m)Tc, ¹⁸⁶Re, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O,⁷⁶Br, and ²¹¹At. Other radionuclides are also available as diagnosticand therapeutic agents, especially those in the energy range of 60 to4,000 keV.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has been reported (Cochlovius et al., Cancer Research (2000)60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (VH1, VL1, VH2, VL2) linked in an orientation to facilitatethe formation of two potential binding sites for each of the twodifferent specificities upon self-association.

Conjugated Anti-CD19 Antibodies

Another embodiment concerns conjugated anti-CD19 antibodies.Compositions and methods for multivalent, multispecific agents aredescribed in U.S. Patent Application Ser. No. 60/436,359, filed Dec. 24,2002, and U.S. Patent Application Ser. No. 60/464,532, filed Apr. 23,2003, which are incorporated herein by reference in their entirety.

Additional amino acid residues may be added to either the N- orC-terminus of the polypeptide. The additional amino acid residues maycomprise a peptide tag, a signal peptide, a cytokine, an enzyme (forexample, a pro-drug activating enzyme), a hormone, a peptide toxin, suchas pseudomonas exotoxin, a peptide drug, a cytotoxic protein or otherfunctional proteins. As used herein, a functional protein is a proteinwhich has a biological function.

In one embodiment, drugs, toxins, radioactive compounds, enzymes,hormones, oligonucleotides, cytotoxic proteins, chelates, cytokines andother functional agents may be conjugated to the target binding protein,preferably through covalent attachments to the side chains of the aminoacid residues of the target binding protein, for example amine,carboxyl, phenyl, thiol or hydroxyl groups. Various conventional linkersmay be used for this purpose, for example, diisocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.Conjugation of agents to the binding protein preferably does notsignificantly affect the protein's binding specificity or affinity toits target. As used herein, a functional agent is an agent which has abiological function. A preferred functional agent is a cytotoxic agent.

As discussed above, enzymes are also useful therapeutic agents. Forexample, alkaline phosphatase for use in combination withphosphate-containing prodrugs (U.S. Pat. No. 4,975,278); arylsulfatasefor use in combination with sulfate-containing prodrugs (U.S. Pat. No.5,270,196); peptidases and proteases, such as serratia protease,thermolysis, subtilisin, carboxypeptidase (U.S. Pat. Nos. 5,660,829;5,587,161; 5,405,990) and cathepsins (including cathepsin B and L), foruse in combination with peptide-based prodrugs;D-alanylcarboxypeptidases for use in combination with D-aminoacid-modified prodrugs; carbohydrate-cleaving enzymes such asbeta-galactosidase and neuraminidase for use in combination withglycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298);beta-lactamase for use in combination with beta-lactam-containingprodrugs; penicillin amidases, such as penicillin-V-amidase (U.S. Pat.No. 4,975,278) or penicillin-G-amidase, for use in combination withdrugs derivatized at their amino nitrogens with phenoxyacetamide orphenylacetamide groups; and cytosine deaminase (U.S. Pat. Nos.5,338,678; 5,545,548) for use in combination with 5-fluorocytosine-basedprodrugs (U.S. Pat. No. 4,975,278), are suitable therapeutic agents.

In still other embodiments, bispecific antibody-directed delivery oftherapeutics or prodrug polymers to in vivo targets can be combined withbispecific antibody delivery of radionuclides, such that combinationchemotherapy and radioimmunotherapy is achieved. Each therapy can beconjugated to the targetable conjugate and administered simultaneously,or the nuclide can be given as part of a first targetable conjugate andthe drug given in a later step as part of a second targetable conjugate.

In another embodiment, cytotoxic agents may be conjugated to a polymericcarrier, and the polymeric carrier may subsequently be conjugated to themultivalent target binding protein. For this method, see Ryser et al.,Proc. Natl. Acad. Sci. USA, 75:3867-3870, 1978, U.S. Pat. No. 4,699,784and U.S. Pat. No. 4,046,722, which are incorporated herein by reference.Conjugation preferably does not significantly affect the bindingspecificity or affinity of the binding protein.

Humanized, Chimeric and Human Antibodies for Treatment and Diagnosis

Humanized, chimeric and human monoclonal antibodies, i.e., anti-CD19MAbs and other MAbs described herein, are suitable for use intherapeutic methods and diagnostic methods. Accordingly, the presentinvention contemplates the administration of the humanized, chimeric andhuman antibodies alone as a naked antibody or administered as amultimodal therapy, temporally according to a dosing regimen, but notconjugated to, a therapeutic agent. The efficacy of the naked anti-CD19MAbs can be enhanced by supplementing naked antibodies with one or moreother naked antibodies, i.e., MAbs to specific antigens, such as CD4,CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37,CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1,Ia, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, an oncogene,an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC,IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) with one or moreimmunoconjugates of anti-CD19, or antibodies to theses recited antigens,conjugated with therapeutic agents, including drugs, toxins,immunomodulators, hormones, enzymes, oligonucleotides, therapeuticradionuclides, etc., with one or more therapeutic agents, includingdrugs, toxins, enzymes, oligonucleotides, immunomodulators, hormones,therapeutic radionuclides, etc., administered concurrently orsequentially or according to a prescribed dosing regimen, with the MAbs.

Preferred B-cell antigens include those equivalent to human CD19, CD20,CD21, CD22, CD23, CD46, CD52, CD74, CD80, and CD5 antigens. PreferredT-cell antigens include those equivalent to human CD4, CD8 and CD25 (theIL-2 receptor) antigens. An equivalent to HLA-DR antigen can be used intreatment of both B-cell and T-cell disorders. Particularly preferredB-cell antigens are those equivalent to human CD19, CD20, CD22, CD21,CD23, CD74, CD80, and HLA-DR antigens. Particularly preferred T-cellantigens are those equivalent to human CD4, CD8 and CD25 antigens. CD46is an antigen on the surface of cancer cells that blockcomplement-dependent lysis (CDC).

Further, the present invention contemplates the administration of animmunoconjugate for diagnostic and therapeutic uses in B cell lymphomasand other disease or disorders. An immunoconjugate, as described herein,is a molecule comprising an antibody component and a therapeutic ordiagnostic agent, including a peptide which may bear the diagnostic ortherapeutic agent. An immunoconjugate retains the immunoreactivity ofthe antibody component, i.e., the antibody moiety has about the same orslightly reduced ability to bind the cognate antigen after conjugationas before conjugation.

A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the claimed antibodies. The therapeuticagents recited here are those agents that also are useful foradministration separately with the naked antibody as described above.Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors,antimitotics, antiangiogenic and apoptotic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, proteosomeinhibitors, and others from these and other classes of anticanceragents, thalidomide and derivates, oligonucleotides, particularlyantisense and RNAi oligonucleotides (e.g., against bc1-2), and the like.Other useful cancer chemotherapeutic drugs for the preparation ofimmunoconjugates and antibody fusion proteins include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, enzymes, hormones, and the like. Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCJhNCES, 19th Ed.(Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), as well as revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated to the claimed antibodies. For example, a therapeuticallyuseful immunoconjugate can be obtained by conjugating a photoactiveagent or dye to an antibody composite. Fluorescent compositions, such asfluorochrome, and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion. In therapy, thishas been termed photoradiation, phototherapy, or photodynamic therapy(Joni et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES(Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986).

Moreover, monoclonal antibodies have been coupled with photoactivateddyes for achieving phototherapy. Mew et al., J. Immunol. 130:1473(1983); idem., Cancer Res. 45:4380 (1985); Oseroff et al., Proc. Natl.Acad. Sci. USA 83:8744 (1986); idem., Photochem. Photobiol. 46:83(1987); Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta etal., Lasers Surg Med. 9:422 (1989); Pelegrin et al., Cancer 67:2529(1991). However, these earlier studies did not include use of endoscopictherapy applications, especially with the use of antibody fragments orsubfragments. Thus, the present invention contemplates the therapeuticuse of immunoconjugates comprising photo active agents or dyes.

Also contemplated by the present invention are the use of radioactiveand non-radioactive agents as diagnostic agents. A suitablenon-radioactive diagnostic agent is a contrast agent suitable formagnetic resonance imaging, computed tomography or ultrasound. Magneticimaging agents include, for example, non-radioactive metals, such asmanganese, iron and gadolinium, complexed with metal-chelatecombinations that include 2-benzyl-DTP A and its monomethyl andcyclohexyl analogs, when used along with the antibodies of theinvention. See U.S. Ser. No. 09/921,290 filed on Oct. 10, 2001, which isincorporated in its entirety by reference.

Furthermore, a radiolabeled antibody or immunoconjugate may comprise agamma-emitting radioisotope or a positron-emitter useful for diagnosticimaging. Suitable radioisotopes, particularly in the energy range of 60to 4,000 keV, include ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ⁶²Cu, ⁶⁴Cu, ¹¹¹In, ⁶⁷Ga,⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, and the like. Seefor example, U.S. Provision al Application No. 60/342,104, whichdiscloses positron emitters, such as ¹⁸F, ⁶⁸Ga, ^(94m)Tc and the like,for imaging purposes and which is incorporated in its entirety byreference.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an antibody fusion protein of ananti-CD19 antibody. Other toxins suitably employed in the preparation ofsuch conjugates or other fusion proteins, include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg, C A—A Cancer Journal for Clinicians 44:43(1994). Additional toxins suitable for use in the present invention areknown to those of skill in the art and are disclosed in U.S. Pat. No.6,077,499, which is incorporated in its entirety by reference.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with the humanized anti-CD19 antibodies. Suitable cytokinesfor the present invention include, but are not limited to, interferonsand interleukins, as described below.

Preparation of Immunoconjugates

Any of the antibodies or antibody fusion proteins can be conjugated withone or more therapeutic or diagnostic agents. Generally, one therapeuticor diagnostic agent is attached to each antibody or antibody fragmentbut more than one therapeutic agent or diagnostic agent can be attachedto the same antibody or antibody fragment. The antibody fusion proteinsmay comprise two or more antibodies or fragments thereof and each of theantibodies that compose this fusion protein can contain a therapeuticagent or diagnostic agent. Additionally, one or more of the antibodiesof the antibody fusion protein can have more than one therapeutic ofdiagnostic agent attached. Further, the therapeutic agents do not needto be the same but can be different therapeutic agents. For example, onecan attach a drug and a radioisotope to the same fusion protein.Particularly, an IgG can be radiolabeled with ¹³¹I and attached to adrug. The ¹³¹I can be incorporated into the tyrosine of the IgG and thedrug attached to the epsilon amino group of the IgG lysines. Boththerapeutic and diagnostic agents also can be attached to reduced SHgroups and to the carbohydrate side chains.

Bispecific antibodies are useful in pretargeting methods and provide apreferred way to deliver therapeutic agents or diagnostic agents to asubject. U.S. Ser. No. 09/382,186 discloses a method of pretargetingusing a bispecific antibody, in which the bispecific antibody is labeledwith ¹²⁵I and delivered to a subject, followed by a divalent peptidelabeled with ^(99m)Tc. Pretargeting methods are also described in U.S.Ser. No. 09/823,746 (Hansen et al.) and Ser. No. 10/150,654 (Goldenberget al.), which are incorporated herein by reference in their entirety.The delivery results in excellent tumor/normal tissue ratios for ¹²⁵Iand ^(99m)Tc. Any combination of known therapeutic agents or diagnosticagents can be used to label the antibodies and antibody fusion proteins.The binding specificity of the antibody component of the MAb conjugate,the efficacy of the therapeutic agent or diagnostic agent and theeffector activity of the Fc portion of the antibody can be determined bystandard testing of the conjugates.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such peptides can be attached to the antibody componentusing a heterobifunctional crosslinker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995).

Alternatively, the therapeutic or diagnostic agent can be conjugated viaa carbohydrate moiety in the Fc region of the antibody. The carbohydrategroup can be used to increase the loading of the same peptide that isbound to a thiol group, or the carbohydrate moiety can be used to bind adifferent peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region is absent if the antibody used as the antibody componentof the immunoconjugate is an antibody fragment. However, it is possibleto introduce a carbohydrate moiety into the light chain variable regionof a full length antibody or antibody fragment. See, for example, Leunget al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incorporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

Pharmaceutically Acceptable Excipients

The humanized, chimeric and human anti-CD19 MAbs to be delivered to asubject can consist of the MAb alone, immunoconjugate, fusion protein,or can comprise one or more pharmaceutically suitable excipients, one ormore additional ingredients, or some combination of these.

The immunoconjugate or naked antibody can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe immunoconjugate or naked antibody are combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The immunoconjugate or naked antibody can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody is administered to a mammal in a therapeutically effectiveamount. A suitable subject is usually a human, although a non-humananimal subject is also contemplated. An antibody preparation is said tobe administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient mammal. In particular, an antibody preparationis physiologically significant if its presence invokes an antitumorresponse or mitigates the signs and symptoms of an autoimmune diseasestate. A physiologically significant effect could also be the evocationof a humoral and/or cellular immune response in the recipient mammal.

Methods of Treatment

The present invention contemplates the use of naked or conjugatedanti-CD19 antibodies as the primary composition for treatment of B celldisorders and other diseases. In particular, the compositions describedherein are particularly useful for treatment of various autoimmunediseases as well as indolent forms of B-cell lymphomas, aggressive formsof B-cell lymphomas, chronic lymphatic leukemias, acute lymphaticleukemias, and Waldenstrom's macroglobulinemia. For example, thehumanized anti-CD19 antibody components and immunoconjugates can be usedto treat both indolent and aggressive forms of non-Hodgkin's lymphoma.

As discussed above, the antibodies are also suitable for diagnosis andtreatment of various autoimmune diseases. Such diseases include acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosurn, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis ubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, pemphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pernicious anemia, rapidly progressive glomerulonephritis, psoriasis,and fibrosing alveolitis. The most common treatments for such diseasesare corticosteroids and cytotoxic drugs, which can be very toxic. Thesedrugs also suppress the entire immune system, can result in seriousinfection, and have adverse effects on the bone marrow, liver andkidneys. Other therapeutics that have been used to treat Class IIIautoimmune diseases to date have been directed against T-cells andmacrophages.

The compositions for treatment contain at least one humanized, chimericor human monoclonal anti-CD19 antibody alone or in combination withother antibodies, such as other humanized, chimeric, or humanantibodies, therapeutic agents or immunomodulators. In particular,combination therapy with a fully human antibody is contemplated.

Naked or conjugated antibodies to the same or different epitope orantigen may also be used in combination. For example, a humanized,chimeric or human naked anti-CD19 antibody may be combined with anothernaked humanized, chimeric or human anti-CD19 or with a naked anti-CD20,anti-CD22 or other B-cell lineage antibody. A humanized, chimeric orhuman naked anti-CD19 antibody may be combined with an anti-CD19immunoconjugate or anti-CD22 radioconjugate. An anti-CD22 naked antibodymay be combined with a humanized, chimeric or human anti-CD19 antibodyconjugated to an isotope, one or more chemotherapeutic agents,cytokines, toxins or a combination thereof. A fusion protein of ahumanized, chimeric or human anti-CD19 antibody and a toxin orimmunomodulator, or a fusion protein of at least two different B-cellantibodies (e.g., an anti-CD19 and an anti-CD22 MAb or an anti-CD19 andan anti-CD20 MAb) may also be used. Many different antibodycombinations, targeting at least two different antigens associated withB-cell disorders, as listed above, may be constructed, either as nakedantibodies or conjugated with a therapeutic agent or immunomodulator, ormerely in combination with another therapeutic agent, such as acytotoxic drug, a cytokine or radionuclide.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-M, IL-12, IL-21 and IL-18), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-alpha, -beta and -gamma), the stem cell growth factordesignated “S1 factor,” erythropoietin and thrombopoietin. Examples ofsuitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12,IL-18, IL-21, interferon, TNF, and the like. Alternatively, subjects canreceive naked anti-CD19 antibodies and a separately administeredcytokine, which can be administered before, concurrently with or afteradministration of the naked anti-CD19 antibodies. As discussed supra,the anti-CD19 antibody may also be conjugated to the immunomodulator.The immunomodulator may also be conjugated to a hybrid antibodyconsisting of one or more antibodies binding to different antigens.

Multimodal therapies further include immunotherapy with naked anti-CD19antibodies supplemented with administration of antibodies that bind CD4,CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37,CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1,Ia, HM1.24, HLA-DR (including the invariant chain), tenascin, VEGF,P1GF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d,necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) andTRAIL-R2 (DR5) in the form of naked antibodies, fusion proteins, or asimmunoconjugates. These antibodies include polyclonal, monoclonal,chimeric, human or humanized antibodies that recognize at least oneepitope on these antigenic determinants. Anti-CD19 and anti-CD22antibodies are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Heiman et al., CancerImmunol. Immunother. 32: 364 (1991); Longo, Curr. Opin. Oncol. 8:353(1996) and U.S. Pat. Nos. 5,798,554 and 6,187,287, incorporated in theirentirety by reference. Immunotherapy of autoimmune disorders with B-cellantibodies is described in the art. See, for example, WO0074718A1, whichis incorporated herein by reference in its entirety.

In another form of multimodal therapy, subjects receive naked anti-CD19antibodies, and/or immunoconjugates, in conjunction with standard cancerchemotherapy. For example, “CVB” (1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen used to treatnon-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51:18 (1993).Other suitable combination chemotherapeutic regimens are well-known tothose of skill in the art. See, for example, Freedman et al.,“Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition,Holland et al. (eds.), pages 2027-2068 (Lea & Febiger 1993). As anillustration, first generation chemotherapeutic regimens for treatmentof intermediate-grade non-Hodgkin's lymphoma (NHL) include C-MOPP(cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone). A usefulsecond generation chemotherapeutic regimen is m-BACOD (methotrexate,bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate and bryostatin-1. Antisense bc1-2 oligonucleotide is also inclinical trials as a therapeutic for certain malignancies, includingB-cell tumors. In a preferred multimodal therapy, both chemotherapeuticdrugs and cytokines are co-administered with an antibody,immunoconjugate or fusion protein. The cytokines, chemotherapeutic drugsand antibody or immunoconjugate can be administered in any order, ortogether.

In a preferred embodiment, NHL is treated with 4 weekly infusions of thehumanized anti-CD19 antibody at a dose of 200-400 mg/m² weekly for 4consecutive weeks (iv over 2-8 hours), repeated as needed over nextmonths/yrs. Also preferred, NHL is treated with 4 weekly infusions asabove, but combined with epratuzumab (anti-CD22 humanized antibody) onthe same days, at a dose of 360 mg/m², given as iv infusion over 1 hour,either before, during or after the anti-CD19 monoclonal antibodyinfusion. Still preferred, NHL is treated with 4 weekly infusions of theanti-CD19 antibody as above, combined with one or more injections ofanti-CD22 MAb radiolabeled with a therapeutic isotope such as yttrium-90(at dose of Y-90 between 5 and 35 mCi/m² as one or more injections overa period of weeks or months. Anti-CD19 MAb may also be combined, insimilar regimens, with anti-CD20 MAbs, such as the hA20 humanized MAb(U.S. application Ser. No. 10/366,709, filed Feb. 14, 2003), whereby aweekly dosex4 weeks per cycle, with optional repeated cycles, is givenof each antibody at an individual dose of 250 mg/m² i.v. in combination.Either or both antibodies can also be given by s.c. injection, whereby asimilar dose is given every other week, particularly for the therapy ofpatients with autoimmune disease.

In addition, a therapeutic composition can contain a mixture or hybridmolecules of monoclonal naked anti-CD19 antibodies directed todifferent, non-blocking CD19 epitopes. Accordingly, the presentinvention contemplates therapeutic compositions comprising a mixture ofmonoclonal anti-CD19 antibodies that bind at least two CD19 epitopes.

Although anti-CD19 antibodies are the primary therapeutic compositionsfor treatment of B cell lymphoma and autoimmune diseases, the efficacyof such antibody therapy can be enhanced by supplementing the nakedantibodies, with supplemental agents, such as immunomodulators, likeinterferons, including IFN-alpha, IFN-beta and IFN-gamma, interleukinsincluding IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IL-21, and cytokinesincluding G-CSF and GM-CSF. Accordingly, the anti-CD19 antibodies can becombined not only with antibodies and cytokines, either as mixtures(given separately or in some predetermined dosing regimen) or asconjugates or fusion proteins to the anti-CD19 antibody, but also can begiven as a combination with drugs. For example, the anti-CD19 antibodymay be combined with CHOP as a 4-drug chemotherapy regimen.Additionally, a naked anti-CD19 antibody may be combined with a nakedanti-CD22 antibody and/or naked anti-CD20 antibodies and CHOP orFludarabine as a drug combination for NHL therapy. The supplementaltherapeutic compositions can be administered before, concurrently orafter administration of the anti-CD19 antibodies.

As discussed supra, the antibodies can be used for treating B celllymphoma and leukemia, and other B cell diseases or disorders. Theantibodies may be used for treating any disease or syndrome whichinvolves unwanted or undesirable B-cell activity or proliferation. Forexample, anti-CD19 antibodies can be used to treat B-cell relatedautoimmune diseases, including Class III autoimmune diseases. Theantibodies can also be used to treat B-cell diseases such as graftversus host disease, or for transplant immunosuppressive therapy.

Anti-CD19 antibodies may also induce apoptosis in cells expressing theCD19 antigen. Evidence of this induction is supported in the literature.For example, it was demonstrated that apoptosis could be induced usinglymphoid cells that have Fc-receptors reactive with the IgG1-Fc ofanti-CD19 MAbs that crosslinked. See Shan et al., Cancer Immunol.Immunother. 48(12):673-683 (2000). Further, it was reported thataggregates of a chimeric anti-CD19 MAb, i.e., homopolymers, inducedapoptosis. See Ghetie et al., Blood 97(5): 1392-1398 (2000) and Ghetieet al., Proc. Natl. Acad. Sci. USA 94(14): 7509-7514 (1997). Enhancementof the pro-apoptotic activity of the antibodies may be achieved bysimultaneous use of a pro-apoptotic agent, such as an agent thatinhibits the activity of one or more members of the anti-apoptosis genefamily bc 1-2. Antisense and RNAi agents are particularly useful in thisregard and can be directed to B cells by conjugation with anti-CD19antibodies as described herein.

Antibodies specific to the CD19 surface antigen of B cells can beinjected into a mammalian subject, which then bind to the CD19 cellsurface antigen of both normal and malignant B cells. A mammaliansubject includes humans and domestic animals, including pets, such asdogs and cats. The anti-CD19 MAbs, i.e., humanized, chimeric, human, andeven murine anti-CD19 MAbs, can be used to treat the non-human mammaliansubjects when there is a species crossreactivity for the CD19 antigen.See Examples 10 and 11, below. The murine MAbs, which are immunogenic inhumans, are usually less immunogenic in non-human mammalian subjects.The anti-CD19 antibody bound to the CD19 surface antigen leads to thedestruction and depletion of neoplastic B cells. Because both normal andmalignant B cells express the CD19 antigen, the anti-CD19 antibody willresult in B cell death. However, only normal B cells will repopulate andthe malignant B cells will be eradicated or significantly reduced.Additionally, chemical agents or radioactive labels having the potentialto destroy the tumor can be conjugated to the anti-CD19 antibody suchthat the agent is specifically targeted to the neoplastic B cells.

Expression Vectors

The DNA sequence encoding a humanized, chimeric or human anti-CD19 MAbcan be recombinantly engineered into a variety of known host vectorsthat provide for replication of the nucleic acid. These vectors can bedesigned, using known methods, to contain the elements necessary fordirecting transcription, translation, or both, of the nucleic acid in acell to which it is delivered. Known methodology can be used to generateexpression constructs that have a protein-coding sequence operablylinked with appropriate transcriptional/translational control signals.These methods include in vitro recombinant DNA techniques and synthetictechniques. For example, see Sambrook et al., 1989, MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel etal., 1997, CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY, John Wiley & Sons(New York).

Vectors suitable for use can be viral or non-viral. Particular examplesof viral vectors include adenovirus, AAV, herpes simplex virus,lentivirus, and retrovirus vectors. An example of a non-viral vector isa plasmid. In a preferred embodiment, the vector is a plasmid.

An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements.

Preferably, the expression vector comprises the DNA sequence encoding ahumanized, chimeric or human anti-CD19 MAb, which includes both theheavy and the light chain variable and constant regions. However, twoexpression vectors may be used, with one comprising the heavy chainvariable and constant regions and the other comprising the light chainvariable and constant regions. Still preferred, the expression vectorfurther comprises a promoter, a DNA sequence encoding a secretion signalpeptide, a genomic sequence encoding a human IgG1 heavy chain constantregion, an Ig enhancer element and at least one DNA sequence encoding aselectable marker.

Also contemplated herein is a method for expressing a humanizedanti-CD19 MAb, comprising (i) linearizing at least one expression vectorcomprising a DNA sequence encoding a humanized, chimeric, or humananti-CD19 MAb, (ii) transfecting mammalian cells with at least one ofsaid linearized vector, (iii) selecting transfected cells which expressa marker gene, and (iv) identifying the cells secreting the humanizedanti-CD19 MAb from the transfected cells.

Methods of Making Anti-CD19 Antibodies

In general, the Vk and VH sequences encoding an anti-CD19 MAb can beobtained by a variety of molecular cloning procedures, such as RT-PCR,5′-RACE, and cDNA library screening. Specifically, the V genes of ananti-CD19 MAb can be cloned by PCR amplification from a cell thatexpresses a murine or chimeric anti-CD19 MAb and then sequenced. Toconfirm their authenticity, the cloned VL and VH genes can be expressedin cell culture as a chimeric Ab as described by Orlandi et al., (Proc.Natl. Acad. Sci., USA, 86: 3833 (1989)) which is incorporated byreference. Based on the V gene sequences, a humanized anti-CD19 MAb canthen be designed and constructed as described by Leung et al. (Mol.Immunol., 32: 1413 (1995)), which is incorporated herein by reference.cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine or chimeric anti-CD19 MAb by general molecularcloning techniques (Sambrook et al., Molecular Cloning, A laboratorymanual, 2nd Ed (1989)). The VK sequence for the MAb may be amplifiedusing the primers Vk1BACK and Vk1FOR (Orlandi et al., 1989) or theextended primer set described by Leung et al. (BioTechniques, 15: 286(1993)), which is incorporated herein by reference, while VH sequencescan be amplified using the primer pair VH1BACK/VH1FOR (Orlandi et al.,1989), or the primers annealing to the constant region of murine IgGdescribed by Leung et al. (Hybridoma, 13:469 (1994)), which isincorporated herein by reference.

The PCR reaction mixtures containing 10 μl of the first strand cDNAproduct, 10 μl of 10× PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3),15 mM MgCl₂, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk,Conn.), 250 μM of each dNTP, 200 nM of the primers, and 5 units of TaqDNA polymerase (Perkin Elmer Cetus) can be subjected to 30 cycles ofPCR. Each PCR cycle preferably consists of denaturation at 94° C. for 1min, annealing at 50° C. for 1.5 mM, and polymerization at 72° C. for1.5 min. Amplified VK and VH fragments can be purified on 2% agarose(BioRad, Richmond, Calif.). Similarly, the humanized V genes can beconstructed by a combination of long oligonucleotide template synthesesand PCR amplification as described by Leung et al. (Mol. Immunol., 32:1413 (1995)). See Example 3 for a method for the synthesis of an oligo Aand an oligo B on an automated RNA/DNA synthesizer (Applied Biosystems,Foster City, Calif.) for use in constructing humanized V genes.

PCR products for VK can be subcloned into a staging vector, such as apBR327-based staging vector, VkpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the VK PCR products. PCR products for VH can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al. (Proc. Natl.Acad. Sci., USA, 74: 5463 (1977)).

The expression cassettes containing the VK and VH, together with thepromoter and signal peptide sequences can be excised from VKpBR andVHpBS, respectively, by double restriction digestion as HindIII-BamHIfragments. The VK and VH expression cassettes can then be ligated intoappropriate expression vectors, such as pKh and pGlg, respectively(Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can beco-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14 orSp-EEE, colonies selected for hygromycin resistance, and supernatantfluids monitored for production of a chimeric or humanized anti-CD19 MAbby, for example, an ELISA assay. Alternatively, the VK and VH expressioncassettes can be assembled in the modified staging vectors, YKpBR2 andVHpBS2, excised as XbaI/BamHI and XhoI/BamHI fragments, respectively,and subcloned into a single expression vector, such as pdHL2, asdescribed by Gilles et al. (J. Immunol. Methods 125:191 (1989)) and alsoshown in Losman et al., (Cancer, 80:2660 (1997)) for expression incells. Another vector that is useful is the GS vector, as described inBarnes et al., Cytotechnology 32: 109-123 (2000), which is preferablyexpressed in the NS0 cell line and CHO cells. Other appropriatemammalian expression systems are described in Werner et al.,Arzneim.-Forsch./Drug Res. 48(II), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VkpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992). Following transfection, cells may be grown in 96-wellmicrotiter plates in complete HSFM medium (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C., 5% CO₂. The selection process can beinitiated after two days by the addition of hygromycin selection medium(Calbiochem, San Diego, Calif.) at a final concentration of 500 units/mlof hygromycin. Colonies typically emerge 2-3 weeks post-electroporation.The cultures can then be expanded for further analysis.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (about. 100 μl) from transfectoma cultures are addedin triplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch) are added to the wells, (100 μl of antibody stockdiluted×10⁴, supplemented with the unconjugated antibody to a finalconcentration of 1.0 μg/ml). Following an incubation of 1 h, the platesare washed, typically three times. A reaction solution, [100 μl,containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 μm membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

The following are the nucleotide sequences of the primers used toprepare the anti-CD19 antibodies:

hA19VkA (SEQ ID NO: 24) 5′-ATCACTTGTA AGGCCAGCCA AAGTGTTGAT TATGATGGTGATAGTTATTT GAACTGGTAC CAGCAGATTC CAGGGAAAGC ACCTAAATTGTTGATCTACG ATGCTTCGAA TCTAGTTTCT GGTATC-3′ hA19VkB (SEQ ID NO: 25)5′-TGCTGACAGT GATATGTTGC AATGTCTTCT GGTTGAAGAGAGCTGATGGT GAAAGTGTAA TCTGTCCCAG ATCCGCTGCC AGAGAATCGAGGAGGGATAC CAGAAACTAG ATTCGAAGCA TCGTA-3  hA19VkBack (SEQ ID NO: 26)5′-TCCGACATCC AGCTGACCCA GTCTCCATCA TCTCTGAGCGCATCTGTTGG AGATAGGGTC ACTATCACTT GTAAGGCCAG CCAAAG-3′ hA19VkFor(SEQ ID NO: 27) 5′-GCTCCTTGAG ATCTGTAGCT TGGTCCCTCC ACCGAACGTCCACGGATCTT CAGTACTTTG CTGACAGTGA TATGTTGCAA-3′ hA19VHA (SEQ ID NO: 28)5′-CTGGCTACGC TTTCAGTAGC TACTGGATGA ACTGGGTGAGGCAGAGGCCT GGACAGGGTC TTGAGTGGAT TGGACAGATT TGGCCTGGAGATGGTGATAC TAACTACAAT GGAAAGTTCA AGGGGCGCGC CACTATT-3′ hA19VHB(SEQ ID NO: 29) 5′-CGTAGTCTCC CGTCTTGCAC AAGAATAGAA CGCTGTGTCCTCAGATCGTA GGCTGCTGAG TTCCATGTAG GCTGTATTAG TGGATTCGTCGGCAGTAATA GTGGCGCGCC CCTTGAACTT TCCATTGTA-3′ hA19VHBack (SEQ ID NO: 30)5′-CAGGTCCAAC TGCAGCAATC AGGGGCTGAA GTCAAGAAACCTGGGTCATCG GTGAAGGTCTC CTGCAAGGCT TCTGGCTACG CTTTCAGTAG C-3′ hA19VHFor(SEQ ID NO: 31) 5′-TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAGTAGTCCATAGCATAGT AATAACGGCC TACCGTCGTA GTCTCCCGTC TTGCACAAG-3′

EXAMPLES

The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples but rather includes all variations that areevident from the teachings provided herein.

Example 1 Construction of a Humanized Anti-CD19 Antibody

A chimeric A19 (cA19) antibody was constructed and expressed in Sp2/0cell. The Vk (SEQ ID NO:1 and SEQ ID NO:3) and VH (SEQ ID NO:3 and SEQID NO:4) sequences of cA19 are shown in FIG. 1. The cA19 antibody wasshown to bind to CD19+ human lymphoma cell lines, such as Raji, Daudi,and Ramos. The Ag-binding specificity of purified cA19 was evaluated bya cell surface competitive binding assay against other anti-CD19antibodies, e.g. B4 (Coulter) and BU12 (Chembiochem). Briefly, varyingconcentrations of cA19 were incubated with Raji cells in the presence ofa constant amount of an I-125 radiolabeled anti-CD19 antibody for 1 h.After washing to remove the unbound antibodies, the cell surface-boundradiolabeled antibody was quantitated by counting the cell pellets in agamma counter. As shown in FIG. 2, cA19 competed with BU12 (Chembiochem)for cell surface binding, indicating these antibodies share similar oroverlapping epitopes of the CD19 molecule.

The light chain and heavy chain variable region sequences encoding thehumanized anti-hCD19 antibody (hA19) were designed and constructed.Comparison of the variable (V) region framework (FR) sequences of thecA19 (FIGS. 1A and 1B) to registered human antibodies in the Kabatdatabase showed that the FRs of cA19 VK exhibited the highest degree ofsequence homology to that of the human antibody REI (VK), while the VHsequence was most closely related with that of the human antibody EU(VH). The VH FR4 sequence of the human antibody NEWM, however, wasbetter aligned with that of cA19 and used to replace the EU FR4 sequencefor the humanization of the A19 heavy chain (FIG. 3B). Therefore, humanREI framework sequences were used for Vk (FIG. 3A), and a combination ofEU and NEWM framework sequences were used for VH (FIG. 3B). There are anumber of amino acid changes in each chain outside of the CDR regionswhen compared to the starting human antibody frameworks. These residuesare 4L, 39I, 58I, 60P, 87H, 100G, and 107K of VK (FIG. 3A) and 5Q, 27Y,28A, 40R, 91S, 94R, 107T, and 108T of VH (FIG. 3B). The DNA and aminoacid sequences of hA19 VK and VH are shown in FIGS. 4A and 4B,respectively.

Example 2 Method of hA19 Antibody Construction

To engineer the CDR-grafted hA19VH and VK genes, a modified strategy asdescribed by Leung et al. (1995) was used to construct the designed VKand VH genes for hA19 using a combination of long oligonucleotidesyntheses and PCR. Briefly, two long synthetic oligonucleotides (ca. 130mer in length) representing the 5′-(sense strand, designated as A) and3′-half (anti-sense strand, designated as B) of a V sequence are used asthe templates in a PCR reaction. The 3′-terminal sequences of the longoligonucleotides A and B are designed to overlap and be complementary toeach other. PCR is initiated by annealing of the 3′-termini of A and Bto form a short double strand DNA flanked by the rest of longoligonucleotides (single strand). Each annealed end serves as a primerfor the replication of the single stranded DNA, resulting in elongationof A and B to form the double-strand DNA. In the presence of two shortoligonucleotide primers, V gene segment is generated by PCRamplification of the double strand DNA.

Heavy Chain

For the construction of hA19 VH domain, the long oligonucleotides,hA19VHA (SEQ ID NO:28, 126-mer) and hA19VHB (SEQ ID NO:29, 128-mer) weresynthesized on an automated DNA synthesizer (Applied Biosystems).hA19VHA represents nt 74 to 126 of the hA19 VH domain, and hA19VHBrepresents the minus strand of the hA19VH domain complementary to nt 178to 306. The 3′-terminal sequences (33 nt residues) of hA19VHA and VHBare complementary to each other. A minimal amount of hA19VHA and VHB(determined empirically) was amplified in the presence of 10 μL of10×PCR Buffer (500 mM KCl, 100 mM Tris-HCl buffer, pH 8.3, 15 mM MgCl₂),2 μmmol of hA19VHBack (5′-CAGGTCCAAC TGCAGCAATC AGGGGCTGAA GTCAAGAAACCTGGGTCATCG GTGAAGGTCTC CTGCAAGGCT TCTGGCTACG CTTTCAGTAG C-3′ SEQ IDNO:30) and hA19VHFor (5′-TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAGTAGTCCATAGCATAGT AATAACGGCC TACCGTCGTA GTCTCCCGTC TTGCACAAG-3′ SEQ ID NO:31),and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk,Conn.). The underlined portions are the restriction sites for subcloningas shown in FIG. 4B. This reaction mixture was subjected to three cyclesof polymerase chain reaction (PCR) consisting of denaturation at 94° C.for 1 minute, annealing at 45° C. for 1 minute, and polymerization at72° C. for 1.5 minutes. This procedure was followed by 27 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at55° C. for 1 minute, and polymerization at 72° C. for 1 minute. Theresulting DNA fragment showed an expected molecular size in agarose gelelectrophoresis. The double-stranded PCR-amplified product for hA19VHwas gel-purified, restriction-digested with PstI and BstEII restrictionenzymes and cloned into the complementary PstI/BstEII restriction sitesof the heavy chain staging vector, VHpBS2, in which the VH sequence wasfully assembled with the DNA sequence encoding the translationinitiation codon and a secretion signal peptide in-frame ligated at the5′-end and an intron sequence at the 3′-end. VHpBS2 is a modifiedstaging vector of VHpBS (Leung et al., Hybridoma, 13:469 (1994)), intowhich a XhoI restriction site was introduced sixteen bases upstream ofthe translation initiation codon to facilitate the next subcloning step.The assembled VH gene was subcloned as a XhoI-BamHI restriction fragmentinto the expression vector, pdHL2, which contains the expressioncassettes for both human IgG heavy and light chains under the control ofIgH enhancer and MT1 promoter, as well as a mouse dhfr gene as a markerfor selection and amplification (FIG. 4B). Since the heavy chain regionof pdHL2 lacks a BamHI restriction site, this ligation requires use of alinker to provide a bridge between the BamHI site of the variable chainand the HindIII site present in the pdHL2 vector. The resultingexpression vector was designated as hAI9VHpdHL2.

For constructing the full length DNA of the humanized VK sequence,hA19VkA (SEQ ID NO:24, 126-mer, represents nt 61 to 186 of the hA19 VKdomain) and hA19VkB (SEQ ID NO:25, 124-mer, represents the minus strandof the hA19 VK domain complementary to nt 157 to 281) were synthesizedas described above. hA19VkA and VkB were amplified by two shortoligonucleotides hA19VkBack (5′-CAGGTCCAAC TGCAGCAATC AGGGGCTGAAGTCAAGAAAC CTGGGTCATCG GTGAAGGTCTC CTGCAAGGCT TCTGGCTACG CTTTCAGTAG C-3′SEQ ID NO:26) and hA19VkFor (5′-TGAGGAGACG GTGACCGTGG TCCCTTGGCCCCAGTAGTCC ATAGCATAGT AATAACGGCC TACCGTCGTA GTCTCCCGTC TTGCACAAG-3′ SEQID NO:27) as described above. The underlined portions are restrictionsites for subcloning as described below. Gel-purified PCR products forhA19 VK were restriction-digested with PvuII and BglII and cloned intothe complementary PvuII/BclI sites of the light chain staging vector,VkpBR2. VkpBR2 is a modified staging vector of VkpBR (Leung et at,Hybridoma, 13:469 (1994)), into which a XbaI restriction site wasintroduced at sixteen bases upstream of the translation initiationcodon. The assembled VK genes were subcloned as XbaI-BamHI restrictionfragments into the expression vector containing the VH sequence,hA19VHpdHL2. The resulting expression vectors were designated ashA19pdHL2.

Example 3 Transfection and Expression of hA19 Antibodies

Approximately 30 μg of the expression vectors for hA19 were linearizedby digestion with Sail and transfected into Sp2/0-Ag14 cells byelectroporation (450V and 25 J-μg). The transfected cells were platedinto 96-well plates for 2 days and then selected for drug-resistance byadding MTX into the medium at a final concentration of 0.075 μM.MTX-resistant colonies emerged in the wells after 2-3 weeks.Supernatants from colonies surviving selection were screened for humanMAb secretion by ELISA assay. Briefly, 100 μl supernatants were addedinto the wells of a microtiter plate precoated with GAH-IgG, F(ab)₂fragment-specific Ab and incubated for 1 h at room temperature. Unboundproteins were removed by washing three times with wash buffer (PBScontaining 0.05% polysorbate 20). HRP-conjugated GAH-IgG, Fcfragment-specific Ab was added to the wells. Following an incubation of1 h, the plate was washed. The bound HRP-conjugated Ab was revealed byreading A490 nm after the addition of a substrate solution containing 4mM OPD and 0.04% H202. Positive cell clones were expanded and hB43 werepurified from cell culture supernatant by affinity chromatography on aProtein A column.

Example 4 Determination of the Antigen-Binding Specificity and Affinityof anti-CD19 Antibodies

The Ag-binding specificity of cA19 and hA19 purified by affinitychromatography on a Protein A column were evaluated and compared by acell surface competitive binding assay. Briefly, a constant amount(100,000 cpm, ˜10 μCi/μg) of ¹²⁵I-labeled cA19 or hA19 was incubatedwith Raji cells in the presence of varying concentrations (0.2-700 nM)of cA19 or hA19 at 4° C. for 1-2 h. Unbound Abs were removed by washingthe cells in PBS. The radioactivity associated with cells was determinedafter washing. As shown in FIG. 2, the purified hA19 competed with¹²⁵I-labeled cA19 for cell surface binding and vice versa, indicatingthe apparent binding avidities are comparable between these two Abs.

The antigen-binding affinity (avidity) constant of hA19 was determinedby direct cell surface binding assay of the radiolabeled Ab andScatchard plot analysis, in comparison to that of cA19. Briefly, hA19and cA19 were labeled with ¹²⁵I by the chloramines-T method. Varyingamounts of either ¹²⁵I-hA19 or ¹²⁵I-cA19 were incubated with 2×10⁵ Rajicells at 4° C. for 2 h and unbound antibodies were removed by washing.The cell-associated radioactivity was counted and Scatchard plotanalysis was performed to determine the maximum number of hA19 and cA19binding sites per cell and the apparent dissociation constants of theequilibrium binding. As shown in FIG. 5, hA19 showed virtually the samebinding affinity as cA19. The apparent dissociation constant values forthese two antibodies were calculated to be 1.1 and 1.2 nM, respectively.

Example 5 Sequence Variants of hA19

The humanized anti-CD19 MAb (hA19) described in Example 2 above wasexpressed in Sp2/0 cells, but the productivity of resulting clones waslow. Numerous transfections (a total of 15) were performed and hundredsof clones were screened. The productivities of positive clones remainedbetween 0.5-3 μg/ml and amplification with methotrexate did not improvethe productivities of subjected clones.

Modifying the Vk Gene Sequence to Reduce the AT Contents

To generate a higher producing hA19 clone, two approaches were used. Thefirst approach (hA19VkpdHL2) was to re-design the hA19Vk gene sequenceto reduce the AT content, which presumably has a negative impact on theAb gene expression. The new hA19Vk gene was synthesized, assembled, andused to reconstruct the expression vector for hA19.

After construction of hA19VkpdHL2 was completed and DNA sequencingconfirmed the sequences to be correct, a maxiprep was performed toprepare the plasmids for transfection. Three transfections wereperformed: #739 (hA19VkpdHL2#1), #740 (hA19VkpdHL2#5) and #741(hB43pdHL2#1). SP-E26 cells and 10 μg of SalI-linearized DNA were used.MTX selection (0.075 μM) started 48 h post electroporation. An ELISA wasused to screen transfections for positive clones. Transfection #739yielded 31 positive clones (13 were selected), #740 yielded 49 positiveclones (13 were selected), and #741 yielded 41 positive clones (19 wereselected). The productivities were determined and monitored by ELISA andranged from 0.5-3 μg/ml, the same as observed for the original hA19construct.

Two serum-free transfections were performed: #746 (hA19VkpdHL2#1) and#747 (hB43pdHL2#1). SP-ESF cells and 10 μg of SalI-linearized DNA wereused for both transfections. MTX selection (0.075 μM) started 48 h postelectroporation. Transfection #746 yielded 9 positive clones and #747yielded 6 positives. The positives were expanded to a 48 well plate andamplified to 0.2 μM MTX. The 45 clones from transfections #739, #740,and #741 were narrowed down to 12 (based on an ELISA reading above 1.0μg/ml) and the MTX was increased to 0.15 μM. The initial p_(max) of theserum-free clones was determined and the productivities were similar tothe clones in serum medium (0.5-1.5 μg/ml).

Two more serum transfections were performed: #756 (hA19VkpdHL2#5) and#757 (hB43pdHL2#1). The transfection conditions were the same as before,however, the slow growing clones were given time to develop to see ifthe p_(m) would be higher. Transfection #756 yielded 19 positive clonesand #757 yielded 8 positive clones. The p_(max) of transfections #756and #757 were compared with transfections #739, #740, #741, #746, and#747. The productivities were similar (˜0.5-3 μg/ml). The best producerswere selected (739.1A9, 740.1B1, and 756.2G7) and amplified to 0.2 μMMTX. Results reported in Table 2 show an average antibody productivityof between 2 and 3 μg/ml.

TABLE 1 Comparison of initial positives and p_(max) values in both 10%FBS and serum-free conditions. Initial p_(max) Positives (ug/ml) SP-E26#739 31 0.5-3 #740 49 0.5-3 #741 41 0.5-3 #756 19 0.5-3 #757 8 0.5-3SP-ESF #746 9  0.5-1.5 #747 6  0.5-1.5

TABLE 2 Average p_(max) and standard deviation values from the threebest hA19VkpdHL2 clones. 0.2 μM Avg. MTX (ug/ml) St. Dev. 739.1A9 2.96±1.40 740.1B1 2.30 ±0.30 756.2G7 2.95 ±0.70

Cell-Based Antibody Dependent Cytotoxicity Assay

To determine whether or not the redesigned vector produced activeantibody, two liters of clone 756.2G7 were purified by protein-Apurification. The purification yielded 7.3 mgs of anti-CD-19 Ab. HPLCand SDS-PAGE showed the purified protein to be pure (not shown). Thepurified protein was used to examine the effects of antibody treatmenton cell proliferation and death. A cell based antibody dependentcytotoxicity assay was performed to compare hA19, hA20, and hLL2 withand without GAH IgG and Fc fragment. Daudi D1-1 and SP-E26 cells wereplated in two 48 well plates at a final density of 150,000 cells/ml.hA19, hA20 and hLL2 were diluted (final concentration of 10 μg/ml) incomplete medium (RPMI for D1-1 and SFM for SP-E26) both with and withoutGAH (final concentration of 40 μg/ml). The Ab mixtures were added to theplate and placed on a shaker for a few minutes and then put in theincubator. An MTT was performed at dayS 3 and 4. The results from day 3showed that there was no difference in cell growth in SP-E26 cells. TheD1-1 cells show inhibition of cell growth in hA19+GAH and hA20+GAH.These results showed that the redesigned hA19 is active.

hA19FpdHL2

The second approach (hA19FpdHL2) was to re-design the hA19VH gene toreplace the heavy chain (VH) framework region amino acid residue serine91 with the consensus phenylalanine residue. The TCT codon for S91 inhA19VHpdHL2 was changed to a TTC codon for F91 in hA19FVHpdHL2. The newhA19F gene was synthesized, assembled, and used to reconstruct theexpression vector for hA19.

After construction of hA19FpdHL2 was completed and DNA sequencingconfirmed the sequences to be correct a maxiprep was performed toprepare the plasmids for transfection. Two transfections were performed:#762 (hA19FpdHL2#2) and #763 (hA19FpdHL2#3). Transfection conditionswere the same as transfections #739, #740, and #741. An ELISA was usedto screen both transfections for positive clones. Transfection #762yielded 13 positive clones (8 were selected), and #743 yielded 18positive clones (16 were selected). The initial productivities weredetermined by ELISA, shown in Table 3.

TABLE 3 Average p_(max) and standard deviation values from thehA19FpdHL2 clones that were selected. 0.075 μM Avg. MTX (ug/ml) St. Dev.762.2D6 42.42 762.2H9 11.34 762.2B10 14.86 762.2D4 12.69 762.2A10 15.96762.2C11 6.34 762.2F11 9.17 763.2B2 11.57 3.39 763.2G2 11.18 1.74763.2B4 9.08 5.27 763.2D4 10.88 13.64 763.2E11 15.33 4.32 763.2C4 11.578.13 763.2B5 2.94 1.13 763.2H5 7.46 5.39 763.2C5 18.91 10.70 763.2H66.54 4.09 763.2A6 14.17 12.64 763.2A9 2.35 763.2A10 2.95 0.95 763.2B1215.76 6.95 763.2C12 6.28 5.50 763.2D3 6.65

Transfection of host cells with the new vector resulted in more than 100positive clones. We randomly picked 30 clones for evaluation. Most ofthese clones were estimated to have yields of antibody production incell culture of between 5-25 mg/L of IgG. This contrasts with the cellculture productivity of the clones generated previously, which were inthe range of 1-2 mg/L. Therefore, we conclude that the substitutionSer91Phe resulted in a significant increase in the expression level ofthe hA19 antibody, with about a 10-fold increase in antibody production.This surprising and unexpected result allows substantially greateramounts of the antibody protein to be produced in cell culture usingexpression in mammalian cell lines.

Example 6 Therapy of Non-Hodgkin's Lymphoma

A patient with indolent, follicular-cell NHL relapses after chemotherapyincluding dexamethasone, and has disease in the chest (para-aortic lymphnodes), an enlarged and involved spleen, and enlarged cervical lymphnodes. The patient is given a course of 300 mg/m² each of hA19 MAbcombined with humanized anti-CD20 MAb (hA20) sequentially on the sameday by i.v. infusion, weekly for 4 weeks, each time being premedicatedwith TYLENOL® and BENADRYL® according to standard, published doses forsuppressing infusion-related reactions. Four weeks later, the patientreturns for the first follow-up examination and the only observation isthat some of the palpable lymph nodes feel softer. Upon returning 3months following the first therapy cycle, the patient's chest diseaseappears to have become reduced by 40% on CT scan, the spleen is abouthalf the pre-therapy size, and the cervical lymph nodes are almost gone.The patient is then given a retreatment cycle, and another three monthslater appears to have a normal-sized spleen, no cervical lymph nodespalpable or measurable on CT scan, and only a small, 1.5-cm lesion inthe chest. The patient continues to appear almost free of disease foranother 4 months.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All of the publications and patent applications and patents cited inthis specification are herein incorporated in their entirety byreference.

1. A bispecific antibody comprising: a) a first anti-CD19 antibody orantigen-binding fragment thereof, wherein the antibody orantigen-binding fragment thereof comprises (i) the light chaincomplementarity determining region CDR sequences CDR1 of SEQ ID NO: 16(KASQSVDYDGDSYLN); CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 of SEQ IDNO: 18 (QQSTEDPWT); (ii) the heavy chain CDR sequences CDR1 of SEQ IDNO: 19 (SYWMN); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3 ofSEQ ID NO: 21 (RETTTVGRYYYAMDY), and (iii) human antibody framework (FR)and constant region sequences with one or more framework region aminoacid residues substituted from the corresponding framework regionsequences of the parent murine antibody, wherein said substituted FRresidues comprise the substitution of serine for phenylalanine at Kabatresidue 91 of the heavy chain variable region; and b) a second antibodyor humanized antibody or antigen-binding fragment thereof.
 2. Thebispecific antibody of claim 1, wherein the anti-CD19 antibody orantigen-binding fragment thereof comprises the sequences of SEQ ID NO:7(hA19VK) and SEQ ID NO:10 (hA19VH).
 3. The bispecific antibody of claim1, wherein the bispecific antibody is an anti-CD19 X anti-CD3 antibody.4. The bispecific antibody of claim 1, wherein the second antibody bindsto an antigen selected from the group consisting of CD3, CD4, CD5, CD8,CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40,CD40L, CD46, CD52, CD54, CD66(a-d), CD74, CD80, CD126, CD138, B7, MUC,Ia, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, an oncogene product,IL-2, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).
 5. The bispecificantibody of claim 1, wherein the first or second antigen-bindingantibody fragment is selected from the group consisting of a F(ab)₂,F(ab′)₂, Fab, Fab′ and scFv antibody fragment.
 6. The bispecificantibody of claim 1, wherein the bispecific antibody is not conjugatedto any therapeutic or diagnostic agent.
 7. The bispecific antibody ofclaim 1, wherein the bispecific antibody is conjugated to at least onetherapeutic or diagnostic agent.
 8. The bispecific antibody of claim 1,wherein said therapeutic agent is selected from the group consisting ofa cytotoxic agent, a radionuclide, an immunomodulator, a hormone, anenzyme, an oligonucleotide and a photoactive therapeutic agent.
 9. Thebispecific antibody of claim 8, wherein said cytotoxic agent is a drugor toxin.
 10. The bispecific antibody of claim 9, wherein said drug isselected from the group consisting of a vinca alkaloid, ananthracycline, a camptothecan, an epipodophyllotoxin, a taxane, aproteosome inhibitor, a nitrogen mustard, an alkyl sulfonate, anitrosourea, an antimetabolite, an alkylating agent, a triazene, a folicacid analog, a COX-2 inhibitor, a pyrimidine analog, a purine analog, aplatinum coordination complex, an antibiotic, a COX-2 inhibitor, ananti-mitotic agent, an anti-angiogenic agent and a pro-apoptotic agent.11. The bispecific antibody of claim 9, wherein said drug is selectedfrom the group consisting of doxorubicin, methotrexate, paclitaxel,cyclophosphamide, etoposide, carmustine, vincristine, procarbazine,prednisone, bleomycin, leucovorin, phenyl butyrate, bryostatin-1 andCPT-11.
 12. The bispecific antibody of claim 9, wherein said toxin isselected from the group consisting of ricin, abrin, alpha toxin,saporin, onconase, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin and Pseudomonas endotoxin.
 13. The bispecificantibody of claim 8, wherein said immunomodulator is selected from thegroup consisting of a cytokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), a stem cell growth factor, erythropoietin andthrombopoietin.
 14. The bispecific antibody of claim 13, wherein saidlymphotoxin is tumor necrosis factor (TNF), said hematopoietic factor isan interleukin (IL), said colony stimulating factor isgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), said interferon isinterferon-alpha, interferon-beta or interferon-gamma, and said stemcell growth factor is S1 factor.
 15. The bispecific antibody of claim 8,wherein said radionuclide is selected from the group consisting of²²⁵Ac, ⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹³¹I, ¹²¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ³²P, ⁶⁴Cu,⁶⁷Cu, ²¹²Bi, ²¹³Bi and ²¹¹At. 16-37. (canceled)